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PubMed1996

A physical and genetic map of the Corynebacterium glutamicum ATCC 13032 chromosome.

A combined physical and genetic map of the Corynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleases SwaI (5'-ATTTAAAT-3'), PacI (5'-TTAATTAA-3'), and PmeI (5'-GTTTAAAC-3') yielding 26,27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacent SwaI fragments, a genomic cosmid library of C.glutamicum was screened for chromosomal inserts containing SwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect the SwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blotted SwaI digests using as probes PacI and PmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partial SwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes to SwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.

Chromosomes, Bacterial↗
PubMed1996

Genome mapping of Clostridium perfringens strains with I-CeuI shows many virulence genes to be plasmid-borne.

The intron-encoded endonuclease I-CeuI from Chlamydomonas eugametos was shown to cleave the circular chromosomes of all Clostridium perfringens strains examined at single sites in the rRNA operons, thereby generating ten fragments suitable for the rapid mapping of virulence genes by pulsed-field gel electrophoresis (PFGE). This method easily distinguishes between plasmid and chromosomal localisations, as I-CeuI only cuts chromosomal DNA. Using this approach, the genes for three of the four typing toxins, beta, epsilon, and tau, in addition to the enterotoxin and lambda-toxin genes, were shown to be plasmid-borne. In a minority of strains, associated with food poisoning, where the enterotoxin toxin gene was located on the chromosome, genes for two of the minor toxins, theta and mu, were missing.

Bacterial Toxins↗
PubMed2002

PCR-based methods for identification of Enterococcus species.

Two DNA-based techniques were used for species identification of enterococci. PvuII digestion of the genus-specific PCR product yielded four different restriction profiles among 20 enterococcal species; one of them was species-specific for E. faecium. In the second case, 32 reference strains belonging to 20 enterococcal species were divided to 12 groups by amplification of internal transcribed spacer of rRNA operon. Interspecies and some intraspecies profile variability was determined. Both methods gave similar results.

DNA, Intergenic↗
PubMed2000

Genetic fingerprinting of Brevibacterium linens by pulsed-field gel electrophoresis and ribotyping.

Members of Brevibacterium linens display physiological features that are relevant for cheese production. The genomes of five B. linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes. Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp. No linear genomic elements were detected. Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes. An EcoRI fragment of 1.4 kb was identified as common to all strains under study. According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B. linens strains here studied.

Blotting, Southern↗
PubMed1997

Construction of a Fibrobacter succinogenes genomic map and demonstration of diversity at the genomic level.

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies.

Animals↗
PubMed2000

Genetic characterization of a new thermotolerant Bacillus licheniformis strain.

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector.

Bacillus↗
PubMed1998

Plastid promoter utilization in a rice embryogenic cell culture.

Plastid promoter utilization was characterized in rice by mapping transcript 5'-ends in samples derived from leaves and cultured embryogenic cells. We have found that rbcL, atpB and the rRNA operon are transcribed by the plastid-encoded plastid RNA polymerase (PEP), while clpP is transcribed by the nucleus-encoded plastid RNA polymerase (NEP) in both chloroplasts and the non-green plastids of embryogenic cultured cells. This finding is in contrast to reports on BY2 tobacco, in which NEP promoter activity in cultured cells was enhanced relative to leaves, facilitating identification of NEP promoters which are undetectable in chloroplasts. Therefore, it appears that activation of plastid NEP promoters in rice is not essential for adaptation to cell culture.

Adenosine Triphosphatases↗
PubMed1998

Viability and gene expression in Chlamydia trachomatis during persistent infection of cultured human monocytes.

The principal host cell for persistently infecting synovial Chlamydia trachomatis is the macrophage. During infection of human monocytes/macrophages in culture this bacterium displays aberrant morphology and produces no new elementary bodies, reflecting the situation in synovium. Here we investigate the metabolic status of C. trachomatis (serovar K) during an extended infection of human peripheral monocytes in vitro. Using reverse transcription-polymerase chain reaction assays, we have shown that primary transcripts from the chlamydial rRNA operons are present throughout a 10-day course of infection. Other assays targeting mRNAs from chlamydial genes encoding r-proteins S5 and L5, the glycyl-tRNA synthetase, the 60-kDa cysteine-rich outer membrane protein, and the KDO transferase indicate that these messengers are also present throughout the entire 10-day period. The gene encoding the 57-kDa heat-shock protein (hsp60) is expressed by the bacterium throughout the 10-day infection of cultured monocytes, but transcript levels from the gene encoding the major outer membrane protein (omp1) appear to be attenuated. Western analyses targeting these latter proteins confirm the presence of the hsp60 gene product, and the virtual absence of major outer membrane protein, in chlamydia-infected cultured human monocytes. Thus, during extended infection of human monocytes in vitro, chlamydia are non-productive but transcriptionally active; the pattern of transcriptional activity reflects that known for persistent C. trachomatis infection in vivo in synovial tissue.

Antibodies, Monoclonal↗
PubMed2006

Enhancement of polyunsaturated fatty acid production by Tn5 transposon in Shewanella baltica.

Transposon Tn5 mutagenesis was used to generate random mutations in Shewanella baltica MAC1, a polyunsaturated fatty acid (PUFA)-producing bacterium. Three mutants produced 3-5 times more eicosapentaenoic acid (EPA 20:5 n-3) compared to the wild type at 10 degrees C. One of the mutants produced 0.3 mg EPA g(-1) when grown at high temperature (30 degrees C). Moreover, 2 mg docosahexaenoic acid (DHA 22:6 n-3) g(-1) was produced by S. baltica mutants at 4 degrees C. Sequencing of insertion mutation(s) showed 96% homology to trimethylamine N-oxide (TMAO) reductase gene and 85% homology to rRNA operons of E. coli. Tn5 transposon mutagenesis therefore is a suitable technique to increase PUFA formation in bacteria.

DNA Transposable Elements↗
PubMed2006

Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth.

We report here the control of potato tuber moth (Phthorimaea operculella) by incorporating a truncated Bacillus thuringiensis cry9Aa2 gene in the plastid genome. Plasmids pSKC84 and pSKC85 are derivatives of a new polycistronic plastid transformation vector, pPRV312L, that carries spectinomycin resistance (aadA) as a selective marker and targets insertions in the trnI-trnA intergenic region. The Cry9Aa2 N-terminal region (82.1 kDa; 734 amino acids) was expressed in a cassette, which consists of 49 nucleotides of the cry9Aa2 leader and the 3'-untranslated region of the plastid rbcL gene (TrbcL), and relies on readthrough transcription from the plastid rRNA operon. In a tobacco leaf bioassay, expression of Cry9Aa2 conferred resistance to potato tuber moth. In accordance, the Cry9Aa2 insecticidal protein accumulated to high levels, approximately 10% of the total soluble cellular protein and approximately 20% in the membrane fraction. However, high-level Cry9Aa2 expression significantly delayed plant development. Thus, a practical system to control potato tuber moth by Cry9Aa2 expression calls for down-regulation of its expression.

Animals↗
PubMed1994

The demonstration by ribotyping of the stability of oral Streptococcus mutans infection over 5 to 7 years in children.

The distribution of serotypes and ribotypes of mutans streptococcal isolates obtained from seven unrelated children at 5 and at 10 or 12 yr of age was investigated. For ribotyping, chromosomal DNA from 5 to 13 isolates per subject was digested with restriction endonucleases EcoRI and HindIII. The DNA fragments were electrophoretically separated, blotted on to nylon membrane and hybridized to the plasmid pKK3535, which contains the rRNA operon of the Escherichia coli chromosome. The ribotypes were unique for each child. In five children only one ribotype and serotype (c, e or f) was found. In one child two serotypes (c and f) were found at baseline and only one (serotype c) in the follow-up sample. In one child the same serotype was not found in the baseline (serotype e) and in the follow-up (serotype c) samples. Every child except one had a ribotype that was identical to one found 5-7 yr later. The results suggest that, at the age of 5 yr, infection by Streptococcus mutans has already stabilized and the colonizing strain remains permanent.

Bacterial Typing Techniques↗
PubMed1996

Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries.

By ribotyping the genetic diversity of mutans streptococci in six 1.5-3-yr-old children with nursing-bottle caries and in six caries-free, age-matched children and in their mothers was examined. The proportion of mutans streptococci in the dental plaque of the children and their levels in the saliva of the mothers were also examined. For ribotyping, chromosomal DNA of isolates obtained from the plaque of the children (3-12 isolates per child) and from the saliva of the mothers (4-13 isolates per mother) was digested with restriction endonuclease HindIII. The DNA fragments were hybridized to the plasmid pKK3535 which contains the rRNA operon of the Escherichia coli chromosome. The results showed that children with nursing-bottle caries exposed to frequent consumption of sucrose had a high proportion of mutans streptococci in plaque and four of them were colonized with more than one ribotype, whereas caries-free children had a low proportion of mutans streptococci in plaque and only one of them harboured more than one ribotype. Mothers of children with nursing bottle caries had similar levels and numbers of ribotypes of mutans streptococci in saliva as the mothers of the caries-free children. In both child groups, mothers were probably the main source of infection with mutans streptococci. Thus, children with nursing-bottle caries were not only heavily infected with mutans streptococci but also often colonized with more than one clonal type. In the child's acquisition of such clones, frequent sugar consumption may have an important role.

Adult↗
PubMed1991

Mycoplasma gallisepticum strain S6 genome contains three regions hybridizing with 16 S rRNA and two regions hybridizing with 23 S and 5 S rRNA.

Southern hybridization and cloning experiments revealed existence of 3 regions hybridizing with 16 S rRNA and 2 regions hybridizing with 23 S and 5 S rRNA in Mycoplasma Gallisepticum strain S6 genome thus forming 4 separate contiguous regions. One set of a putative rRNA genes is organized classically for eubacteria order 16 S-23 S-5 S. The other two 16 S rRNA and the other one 23 S-5 S rRNA hybridizing regions are separated from each other and from the complete rRNA operon for a distance of more than 6 kb.

Base Sequence↗
PubMed1990

The excision of intervening sequences from Salmonella 23S ribosomal RNA.

Novel, approximately 90 bp intervening sequences (IVs) were discovered within the 23S rRNA genes of S. typhimurium and S. arizonae. These non-rRNA sequences are transcribed and then excised during rRNA maturation. The rRNA fragments that result from the excision of the extra sequences are not religated. This results in fragmented 23S rRNAs. The excision of one IVS was shown to be catalyzed in vivo and in vitro by ribonuclease III. These IVSs are highly volatile evolutionarily, sometimes occurring in only some of the multiple rRNA operons of a particular cell. The sporadic nature of the occurrence of fragmented rRNAs among closely related organisms argues that such fragmentation is a derived state, not a primitive one. Possible sources of these IVSs, their parallels with internal transcribed spacers and introns in eukaryotes, and their possible roles in the evolutionary process are discussed.

Base Sequence↗
PubMed1994

Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation.

The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis (amiB), DNA repair (mutL), tRNA modification (miaA) and proteolysis (hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutL and miaA. In addition, two internal promoters, PmiaA and P1hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. PmiaA contains poor matches to the -10 and -35 regions of the sigma-70 RNA polymerase consensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, PmiaA transcript and P1hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne+ parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc+ parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinate mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.

Amino Acid Sequence↗
PubMed1984

Plasmid vectors for the selection of promoters.

Vectors were constructed which contain promoterless genes for chloramphenicol (cam) or tetracycline (tet) resistance, as promoter-probe plasmids. Escherichia coli cells harboring these plasmids are sensitive to cam or tet but resistant to ampicillin. In plasmids pKK231 -1 and pKK232 -8 the gene for cam acetyltransferase (CAT) and in pKK175 -6 the gene for tet resistance are flanked by efficient transcription terminators, preventing transcription from other pBR322 promoters into the antibiotic resistance region. In one of the vectors, pKK232 -8, translational stop codons were introduced in all three reading frames upstream from the initiation codon of the cat gene. If a DNA fragment containing a promoter is inserted into one of the cloning sites upstream from the antibiotic genes, cells carrying such plasmids acquire resistance to cam or tet. Using these vectors two restriction fragments that contain promoters were identified. One of these fragments contains sequences upstream from an unidentified gene ( ORFII ) located distal to the rrnB rRNA operon of E. coli.

Acetyltransferases↗
PubMed1991

Identification and characterization of the ribosomal RNA-encoding genes in Clavibacter xyli subsp. cynodontis.

Clavibacter xyli subsp. cynodontis (Cxc) is a xylem-inhabiting bacterial endophyte of Bermudagrass. This organism is classified with Gram-positive, high G + C content, coryneform-actinomycete bacteria. Southern-blot analysis showed that Cxc contains only one copy of the ribosomal RNA-encoding genes (rRNA). A clone containing the rRNA genes was isolated from a genomic library of Cxc DNA cloned in the lambda EMBL3 vector. The gene cluster was partially sequenced, revealing the gene order 5'-16S-23S-5S-3', similar to that found in other prokaryotes. Low-resolution S1 mapping suggested multiple transcription start points of the rRNA operon.

Actinomycetales↗
PubMed1994

Characterization of the 16S-23S rRNA intergenic spacer of Bartonella bacilliformis.

The 16S-23S intergenic spacer region from the ribosomal RNA (rRNA) operon of Bartonella bacilliformis was cloned and characterized. The spacer is 906 nucleotides (nt) in length and contains the genes encoding isoleucine-tRNA (tRNA(Ile)) and alanine-tRNA (tRNA(Ala)). The tRNA-encoding genes are separated by 122 nt and are centrally located in the 16S-23S spacer region, with approx. 300 flanking nt. Genes encoding tRNA(Ile) and tRNA(Ala) have 88.3 and 93.4% sequence identity, respectively, to the homologous genes of Rhodobacter sphaeroides.

Bartonella↗