Biomedical subjects

M Romantschuk

Publications and source records attributed to M Romantschuk.

At least 37 records · Page 2Linked to original sources

PubMed1998

Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000.

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.

Amino Acid Sequence↗

PubMed1998

Characterization and reclassification of an aromatic- and chloroaromatic-degrading Pseudomonas sp., strain HV3, as Sphingomonas sp. HV3.

Phylogenetic analyses of 16S rRNA gene sequences showed that the Gram-negative aromatic- and chloroaromatic-degrading Pseudomonas sp. strain HV3 carrying the mega-plasmid pSKY4 belongs to the genus Sphingomonas. The 16SrRNA sequence is most related to Sphingomonas chlorophenolica strains ATCC 33790(T) (98.5%) and SR3 (98.4%) and Sphingomonas sp. SS86 (98.4%). The G+C content was 64 mol%, and the DNA-DNA hybridization-based relative homology of strain HV3 to the S. chlorophenolica ATCC 33790(T) and S. chlorophenolica RA2 was 59.6% and 35.9%, respectively. The results showed that although strain HV3 is related to S. chlorophenolica it differs in certain characteristics. It is therefore proposed to reclassify Pseudomonas sp. strain HV3 as Sphingomonas sp. HV3.

Base Sequence↗

PubMed1997

Purified HrpA of Pseudomonas syringae pv. tomato DC3000 reassembles into pili.

Pseudomonas syringae pv. tomato DC3000 produces Hrp pili under inducing in vitro conditions. A preparation of partially purified extracellular filaments contains HrpA, flagellin and some minor contaminants. HrpA was separated from the major contaminant, the flagellin, by gel filtration to a fraction containing HrpA as well as its three N-terminally truncated forms. These were further separated by two steps of reversed phase chromatography. HrpA and its degradation products were each shown to reassemble into filament structures after denaturation and renaturation showing that HrpA alone is sufficient for formation of filament structures.

Amino Acid Sequence↗

PubMed1997

Novel organization of catechol meta-pathway genes in Sphingomonas sp. HV3 pSKY4 plasmid.

Sphingomonas sp. strain HV3 (formerly Pseudomonas sp. HV3), which degrades aromatics and chloroaromatics, harbors a mega-plasmid, pSKY4. A sequenced 4 kb fragment of the plasmid reveals a novel gene organization for catechol meta-pathway genes. The putative meta operon starts with the cmpF gene encoding a 2-hydroxymuconic semialdehyde hydrolase. The gene has a 6 bp overlap with the previously characterized ring-cleavage gene, catechol 2,3-dioxygenase, cmpE. Downstream of cmpE is a 429 bp open reading frame of unknown function. Gene cmpC, encoding a 2-hydroxymuconic semialdehyde dehydrogenase, starts 44 bp further downstream. It has the highest homology to 2-hydroxymuconic semialdehyde dehydrogenases of dmp and xyl pathways and to XylC from the marine oligotroph Cycloclasticus oligotrophus. The gene organization is different from other known meta pathways. This is the first report of organization of plasmid-encoded meta-pathway genes in the genus Sphingomonas.

Amino Acid Sequence↗

PubMed1997

Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000.

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.

Bacterial Proteins↗

PubMed1996

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

Bacteria↗

PubMed1996

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.

Amino Acid Sequence↗

PubMed1995

Repeated sequences isolated from Bordetella pertussis induce DNA rearrangements and deletions at high frequency.

Two repeated sequences (RS) from Bordetella pertussis were cloned in Escherichia coli and sequenced. The RS, called RSBP1 and RSBP3, are highly homologous to other B. pertussis RS. The recombinant plasmids containing RSBP1 and RSBP3 or transposon-like structures of these elements were not stable but segregated plasmids with deletions or rearranged DNA. RS of B. pertussis seem to be able to stimulate both intra- and inter-genomic RecA-independent recombination events. In at least one case, the observed deletion had occurred precisely between the RS terminus and a site with sequence homology to the terminus. The high frequency rearrangements associated with the RS imply that the RS are transposable elements.

Base Sequence↗

PubMed1995

High frequency of conjugation versus plasmid segregation of RP1 in epiphytic Pseudomonas syringae populations.

The maintenance and transfer of the broad host-range plasmid RP1 in epiphytically growing populations of Pseudomonas syringae was monitored in the phyllosphere of bush bean (Phaseolus vulgaris). When foliage was inoculated with plasmid-containing bacteria, the plasmid was lost from the majority of the cells within 2 d but was stably maintained in 0.8% of the population. A high frequency of conjugation between added donors and recipients was observed under high humidity conditions. In 1 d, the number of transconjugants rose to 10(-1) of the donors and the proportional level of transconjugants continued to increase until 3 d after inoculation. Under these conditions the proportion of plasmid-containing bacteria stabilized at about 0.8% of the total population. The conjugation rate appeared to be in equilibrium with plasmid loss and the slower growth of the plasmid-carrying cells. A factor that influenced the high conjugation frequency observed was the available nutrients provided by the leaf and also, to a lesser extent, the leaf surface itself. Transfer of the plasmid from added donors to indigenous bacteria was also studied, using a donor-specific bacteriophage for counterselection of the donor. Transfer was observed to 10 different species of Gram-negative epiphytically growing bacteria. The bean leaf surface appears to function as a hotspot at least for intraspecific transfer of plasmids in high humidity. The frequency of transfer was higher than in soil or in rhizosphere habitats. This is likely to be the result of an environment that is nutritionally rich in combination with a limited colonizable surface area which permits close contact between the bacterial cells.

Conjugation, Genetic↗

PubMed1994

Cloning of cmpE, a plasmid-borne catechol 2,3-dioxygenase-encoding gene from the aromatic- and chloroaromatic-degrading Pseudomonas sp. HV3.

Pseudomonas sp. strain HV3 degrades aromatics and chloroaromatics. It harbours a mega-plasmid, designated pSKY4, from which the gene cmpE, encoding a catechol 2,3-dioxygenase (C23O) catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde, was cloned and sequenced. The deduced amino acid (aa) sequence shows the highest homology, 52%, to the deduced aa sequences of xylE1 and dmpB. The deduced 307-aa sequence of cmpE contains the extradiol ring-cleavage signature in the same position as other 307-aa C23O-encoding genes.

Amino Acid Sequence↗

PubMed1991

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola.

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.

Amino Acid Sequence↗

PubMed1990

Purified phi 6 nucleocapsids are capable of productive infection of host cells with partially disrupted outer membranes.

Purified nucleocapsids of bacteriophage phi 6, lacking the phage lipid envelope, are unable to infect intact Pseudomonas syringae host cells. A method for studying the process by which a naked virus particle, the phi 6 nucleocapsid, penetrates the host cytoplasmic membrane was developed. Host cells were rendered competent for nucleocapsid infection by treatment with repeated washings with salt and sucrose and the subsequent addition of lysozyme. This treatment disrupts the outer membrane, permitting the nucleocapsid to reach the cytoplasmic membrane and to infect the cell. The nucleocapsid infection is blocked by monoclonal antibodies raised against the nucleocapsid shell protein P8.

Absorption↗

PubMed1988

The nucleocapsid of bacteriophage phi 6 penetrates the host cytoplasmic membrane.

Bacteriophage phi 6 infects its host, the Gram-negative bacterium Pseudomonas syringae, by a protein-targeted fusion of the virus envelope with the host outer membrane. In this investigation we present results suggesting that the phage nucleocapsid penetrates the host cytoplasmic membrane via a membrane invagination and an intracellular vesicle. This indicates that the prokaryotic plasma membrane might be more dynamic and have more common features with eukaryotic membrane systems than previously expected. Most of the nucleocapsid surface lattice protein is degraded in the cell, and the nucleocapsid core particle containing the viral dsRNA segments and the proteins necessary for the viral RNA polymerase activity can be isolated from the infected cells. The penetration is dependent on the energized state of the host cytoplasmic membrane. About 25% of the entering core particles are re-used in the progeny viruses.

Bacteriophages↗

PubMed1988

Nucleotide sequence of the middle dsRNA segment of bacteriophage phi 6: placement of the genes of membrane-associated proteins.

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We have determined the nucleotide sequence of cDNA derived from the middle-size RNA segment. The coding sequences of three proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through the analysis of isolated proteins. In contrast to our results with the small phi 6 dsRNA segment, the open reading frames are not tightly clustered. The homologous terminal noncoding regions between the middle and small dsRNA segments are found to be more extensive than RNA sequencing had previously indicated.

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of the large double-stranded RNA segment of bacteriophage phi 6: genes specifying the viral replicase and transcriptase.

The genome of the lipid-containing bacteriophage phi 6 contains three segments of double-stranded RNA. We determined the nucleotide sequence of cDNA derived from the largest RNA segment (L). This segment specifies the procapsid proteins necessary for transcription and replication of the phi 6 genome. The coding sequences of the four proteins on this segment were identified on the basis of size and the correlation of predicted N-terminal amino acid sequences with those found through analysis of isolated proteins. This report completes the sequence analysis of phi 6. This constitutes the first complete sequence of a double-stranded RNA genome virus.

Amino Acid Sequence↗

PubMed1987

Membrane fusion in prokaryotes: bacteriophage phi 6 membrane fuses with the Pseudomonas syringae outer membrane.

Protein-triggered membrane fusion in the prokaryotic system is described using the lipid-containing enveloped bacterial virus phi 6 and its host, the Gram-negative bacterium Pseudomonas syringae. Bacteriophage particles can be fused to form multiple particles where two or more nucleocapsids are surrounded by a single membrane vesicle with a volume proportional to the number of fused particles. For fusion to occur, a fusogenic protein is required in the membrane of the participating phage particles. Upon infection of the host cell, fusion of the viral membrane with the bacterial membrane takes place without leakage of the periplasmic enzyme alkaline phosphatase to the extracellular supernatant. There is a time-dependent mixing of fluorescent phage phospholipids with the bacterial membrane lipids between 5 and 20 min post-infection. The phage membrane proteins and phospholipids co-purify with the bacterial outer membrane of infected cells. The fusion is independent of divalent cations and pH, resembling Sendai virus fusion with the plasma membrane. This is the first targeted, protein-dependent fusion event described in prokaryotes.

Alkaline Phosphatase↗

PubMed1986

The causal agent of halo blight in bean, Pseudomonas syringae pv. phaseolicola, attaches to stomata via its pili.

The phytopathogenic pseudomonad Pseudomonas syringae pv. phaseolicola causes halo blight of bean (Phaseolus vulgaris L.). Initiation of infection depends on the ability of the cells to adhere to the target cell surface. P. syringae pv. phaseolicola expresses pili, which are the receptors of the lipid-containing dsRNA bacteriophage phi 6. phi 6-resistant bacterial strains can be divided into different piliation types. It was possible to show that the adhesion of the bacteria onto plant cell surface was dependent on the pili. Non-piliated bacterial stains showed a much lower adherence to the leaf surface than strains expressing phi 6 specific pili. Scanning electron microscopy showed that the piliated bacteria attached to the leaf surface at the site of stomata. Non-piliated bacteria were evenly distributed on the leaf surface. All bacterial strains used in this study were capable of causing halo blight if injected into the plant. If the bacteria were sprayed on the plants, followed by spraying of sterile buffer, only piliated bacteria caused symptoms.

Bacterial Adhesion↗

PubMed1985

Function of pili in bacteriophage phi 6 penetration.

The genome of bacteriophage phi 6, which has a lipid protein envelope, consists of three pieces of dsRNA. Virus infection is initiated by attachment to a phi 6-specific host pilus followed by fusion of the phage membrane and the bacterial outer membrane. In this study we analysed several different phi 6 hosts as well as more than 200 independently isolated phi 6-resistant variants derived from Pseudomonas syringae pv. phaseolicola. It is shown that phi 6-specific pili are coded by genes located in the host chromosome. It appears that pilus reaction is needed to pull the pilus-associated virus through the extracellular polysaccharide of the host and thus to bring it into contact with the outer membrane where membrane fusion can take place.

Adsorption↗