Genome analysis ofPropionibacterium freudenreichii by pulsed-field gel electrophoresis

Preparations of intact genomic DNA from 23 strains ofPropionibacterium freudenreichii were compared by digestion with restriction endonucleases and subsequent transverse alternating field electrophoresis (TAFE). Seven restriction enzymes,AsnI,DraI,HpaI,SnaBI,SpeI,SspI, andXbaI, produced DNA fragments useful for strain comparisons. A characteristic restriction fragment pattern was identified for 18 of the 23 strains. Estimates for the genome size of theP. freudenreichii strains ranged from 1.6×10⁶ to 2.3×10⁶ base pairs based on the sum of fragment sizes obtained with restriction digests. Restriction endonuclease patterns resolved by TAFE are useful for strain identification.     

Purification and characterization of endonucleases DraII and III from Deinococcus radiophilus

Deinococcus radiophilus strain ATCC 27603 contains, apart from endonuclease DraI [1], two additional sequence-specific endonucleases. These enzymes, designated DraII and DraIII, recognize nucleotide sequences with novel specificities, PuG↓GNCCPy and CACNN↓GTG, respectively.     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.     

Sizing of theRhodococcus sp. R312 genome by pulsed-field gel electrophoresis. Localization of genes involved in nitrile degradation

The two restriction enzymesAsnI andDraI were found to produce DNA fragment sizes that could be used for mapping theRhodococcus sp. R312 (formerlyBrevibacterium sp. R312) genome by pulsed-field gel electrophoresis.AsnI produced 24 fragments (4 to 727 kb) andDraI yielded 15 fragments (8.5 to 2400 kb). The fragment lengths in each digest were summed, indicating that the size of the chromosome ranged from 6.31 to 6.56 Mb, with a mean of 6.44 Mb. In addition, the wide-spectrum amidase gene (amiE) and the operon containing the enantiomer-selective amidase gene (amdA) and the nitrile hydratase structural gene (nthA, nthB) were localized on theAsnI andDraI fragments.     

Neither Indole Acetic Acid nor Bacteriocin is Apparently Involved in the in vitro Antagonism between the Virulent and the Avirulent Strains of Pseudomonas solanacearum

An avirulent strain of Pseudomonas solanacearum could inhibit the growth of its virulent parent on L-tryptophan-containing glycerol nutrient agar (TGNA) medium. It was, also, capable of inhibiting, though to a less degree, Corynebacterium fascians and Pseudomonas marginata, out of five other bacterial species tested. While P. marginata was partially inhibited by the avirulent strain it was totally insensitive to indole-3-acetic acid (IAA) up to a concentration of 300 μg/ml. Additionally, Erwinia carotovora var. atroseptica which was totally unaffected by the avirulent strain showed a spectrum of sensitivity to IAA concentrations close to that of the virulent strain. No DNA, RNA, or IAA could ever be detected in the inhibition area and, thus, it is almost certain that the inhibiting agent produced by the avirulent strain is not IAA as was previously speculated. This inhibiting agent was insensitive to autoclaving and to the enzymes, pronase, trypsin, DNAse, and RNAse. P. solanacearum bacteriocin was detected by polyacrylamide gel electrophoresis in the medium near the avirulent growth line and not throughout the inhibition area. This supports the conclusion that bacteriocin alone cannot be held responsible for the inhibition phenomenon observed and leaves the nature of this inhibiting agent unknown.     

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10⁵/μg of DNA to 10⁷/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.     

<Emphasis Type="Italic">Rhodococcus fascians</Emphasis>: Shoot Proliferation without Elevated Cytokinins?

In order to determine whether the disease symptoms caused by virulent strains of Rhodococcus fascians are due to increased cytokinin activity in infected tissues, germinating peas (Pisum sativum cv Novella) were inoculated with either a virulent strain or a nonvirulent strain of Rhodococcus fascians. The nonvirulent strain lacked both the ipt gene and the putative cytokinin oxidase/dehydrogenase homologue, fas5. Control peas were not inoculated. Twelve cytokinins were isolated from pea shoots 3, 6 and 9 days post-inoculation. Within 6 days of inoculation the levels of cytokinin free bases, ribosides, O-glucosides and nucleotides were decreased in shoots inoculated with the virulent strain, and were increased in shoots inoculated with the nonvirulent strain relative to the uninoculated control. The results are discussed with respect to the classic Skoog and Miller (1965) model of organogenesis and to the possible involvement of the plant cytokinin oxidase/dehydrogenase during infection by virulent strains of R. fascians.     

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. Received: 13 January 2000 / Accepted: 9 February 2000     

Isolation of some strains ofCorynebacterium fascians (Tilford) Dowson in Czechoslovakia

Four strains of the phytopathogenic bacteriumCorynebacterium fascians (Tilford)Dowson were selected from our isolates and deposited in the Czechoslovak National Collection of Type Cultures (CNCTC) of the Institute of Hygiene and Epidemiology in Prague. Two very virulent (Cor 83/82 “UPR” and Cor 82/81 “UP”) and one avirulent (Cor 81/80 “CP1b”) pelargonium strains, producing acid from rhamnose, were isolated from fasciations onPelargonium zonale W. One avirulent celery strain ofC. fascians (Cor 80/80 “CF4a”) was isolated from a root expiant ofApium graveolens L. growing on a nutrient mediumin vitro. Morphological, cultural, physiological and biochemical characteristics of these selected Czechoslovak isolates were compared with the American patented strain cotype ATCC 12974.     

A large inversion in the linear chromosome of <Emphasis Type="Italic">Streptomyces griseus</Emphasis> caused by replicative transposition of a new Tn<Emphasis Type="Italic">3</Emphasis> family transposon

We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.     

Characterization of the rrnB Operon of the Plant Pathogen Rhodococcus fascians and Targeted Integrations of Exogenous Genes at rrn Loci

A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5′ and 3′ ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (= NRRL {"type":"entrez-nucleotide","attrs":{"text":"B15096","term_id":"2122845","term_text":"B15096"}}B15096), also classified as R. fascians, produced five rrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

Rhodococcus fascians is a gram-positive bacterium with a high G+C content belonging to the group of lower actinomycetes (14) closely related to corynebacteria. Strains of this species are of interest because they are phytopathogenic (32), causing the formation of galls on dicotyledonous plants (30) and malformations of bulbs of monocotyledonous plants (24).The molecular genetics of nonpathogenic corynebacteria have received considerable attention (for reviews see references 23 and 29), but there are no advanced recombinant DNA tools for studying molecular genetics of plant-pathogenic bacteria such as R. fascians. Several plasmids, including circular and high-molecular-weight linear plasmids, are present in strains of R. fascians (7, 11). Conjugative plasmids carrying genes determining resistance to cadmium salts (10) or chloramphenicol (12) have been characterized. One of these plasmids, pRF2, was used to develop bifunctional vectors that also replicate in Escherichia coli (12). By using these vectors, transformation of R. fascians strains has been obtained by electroporation (9, 11).Some genes associated with phytopathogenicity were found in a 200-kb linear plasmid in R. fascians D188 (7, 8). Chromosomal genes also appear to be required to produce plant disease (7). In order to clone and study additional genetic determinants involved in plant pathogenicity, there is a need to develop a system for chromosomal integration and expression of homologous or heterologous DNA in well-characterized dispensable sites of the R. fascians chromosome. As part of an effort in this direction, we characterized the nucleotide sequence and organization of an rRNA operon (rrnB) of R. fascians D188. Although the 5S rRNA gene of this species (21) and the 16S rRNA gene were amplified by PCR and used in phylogenetic analyses of gram-positive bacteria previously (26), the complete organization of the rrn operons and the number of rrn loci were not established.Gene targeting is a useful strategy for introducing exogenous genes into specific chromosomal regions. Due to their repetitive nature, rRNA operons are very suitable targets for chromosomal integration of foreign DNA fragments without modification of growth rates and viability characteristics (5). In this paper we describe the characterization of the rrnB locus and the use of rRNA-encoding regions of R. fascians D188 as target sites for integration and expression of the exogenous gene cmr, a gene conferring chloramphenicol resistance (12).     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ⁻ mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome. Received: 29 July 1996 / Accepted: 27 August 1996     

A 78-megadalton plasmid occurs in avirulent strains as well as virulent strains ofCorynebacterium fascians

Ten wild-type strains ofCorynebacterium fascians, which differed in degree of virulence as measured by ability to cause hyperplasias (multiple stems; fasciation) in pea seedlings, were examined for the presence of plasmids. Four strains were highly virulent, three were avirulent, and three were intermediate in virulence. All of these wild-type strains harbored one plasmid each of approximately 78 megadaltons, as estimated from electrophoretic mobilities in agarose gels capable of resolving reference plasmids ranging from 8.8 to 350 Mdal. Restriction endonuclease (EcoRI andBamHI) cleavage patterns of these nominally 78-MdalC. fascians plasmids suggest that the plasmids are not uniformly of high homology, although similar or identical in electrophoretic mobility in the system used. The relationship of the 78-Mdal plasmids to the phytopathogenicity ofC. fascians remains uncertain, although the restriction endonuclease cleavage patterns do indicate that the plasmids from the highly virulent strains are more closely related to the plasmids from the strains intermediate in virulence than they are to the plasmids from the avirulent strains.     

The plant pathogen Rhodococcus fascians. History,disease symptomatology,host range,pathogenesis and plant–pathogen interaction

Since 1927, outbreaks of the leafy gall disease caused by Rhodococcus fascians on annual and perennial herbaceous plants have put the ornamental plant industry at risk. More recently, Pistachio Bushy top syndrome (PBTS), a serious disease emerging in the Western United States, has been declared as a natural disaster by the U.S. Department of Agriculture. Recent developments indicate that the taxonomic position of R. fascians should be re-evaluated and circumscribed from other members of the genus. Due to the broad host range of this bacterium, economically important crops are in a vulnerable position especially with new members within the genus becoming able to interact synergistically with R. fascians to cause symptom development. The virulence strategies employed by R. fascians and the involvement of cytokinins (CKs) in its pathogenicity have been the subject of much controversy in the last years. Additionally, the detection of virulent strains of R. fascians from symptomatic tissues has been problematic for many researchers. This review focuses on plant–pathogen interaction in context with more recent findings on the association of CKs in virulence of R. fascians. Current knowledge on the worldwide distribution of the pathogen, a historical recap of its discovery and the economic impact of the disease caused by R. fascians are also described here.     

Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ^? mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are discussed.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.     

The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.     

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25 Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region. Received: May 6, 1998 / Accepted: May 26, 1998     

Isolation and characterization of plasmid from the Bacillus brevis var. G.-B. cells

Summary The plasmid designated pAD1 was isolated from the cells of four variants of Bacillus brevis var. G.-B. The plasmid DNA has a molecular weight of about 47.1×10⁶ daltons and contains 43.4 mole % G+C. The bulk of pAD1 DNA (96–98%) is associated with the fraction of chromosome DNA and membranes.Restriction endonucleases SmaI, SalI and BamHI cleaved the plasmid DNA into two, two and six fragments, respectively. The cleavage map of the pAD1 genome has been constructed for these three endonucleases. Restriction enzymes EcoRI, HindIII, KpnI and PstI hydrolized the plasmid DNA into 16, 21, 10 and 9 fragments, respectively. The presence of repeated sequences in the plasmid genome was shown based on pAD1 DNA cleavage by these endonucleases.