Search PubMed⌕ Search

Biomedical subjects

A M Kropinski

Publications and source records attributed to A M Kropinski.

At least 19 recordsLinked to original sources
PubMed1997

Overexpression, purification, and analysis of the c1 repressor protein of Pseudomonas aeruginosa bacteriophage D3.

A 3.1-kb region of the bacteriophage D3 genome which contains the immunity functions has recently been sequenced (GenBank accession No. L22692). Sequence analysis indicated the presence of a putative repressor gene (c1) whose protein product functions to maintain the bacteriophage genome as a stably integrated prophage in the chromosome of Pseudomonas aeruginosa. A plasmid was constructed that overexpresses repressor C1 protein under control of P(tac) in Escherichia coli. C1 protein was subsequently purified and characterized as a 223 amino acid protein with specific binding affinity for 14-base imperfect palindromic operator sequences located on the genome of bacteriophage D3. N-terminal protein sequence data obtained from automated Edman degradation (16 cycles) of purified repressor protein were identical to the predicted sequence based on DNA sequence analysis of the c1 open reading frame.

Amino Acid Sequence↗
PubMed1997

Functional analysis of sigma-70 consensus promoters in Pseudomonas aeruginosa and Escherichia coli.

A series of synthetic promoters, based upon the Escherichia coli sigma 70 consensus promoter sequence, was constructed upstream of the lacZ reporter gene in the modified broad-host-range vector pQF52. The role of the intervening spacer region in gene expression in Pseudomonas aeruginosa and E. coli was studied by insertions and deletions within this region. In P. aeruginosa and E. coli the patterns of gene expression were identical with maximum beta-galactosidase activity being measured from promoters possessing 19 bp in their intervening regions, presumably as a result of impeded promoter clearance with the consensus 17-bp promoter. In P. aeruginosa a second occurrence of enhanced activity, which could not be attributed to the involvement of the alternative sigma factor RpoN (sigma 54), was evident with the promoter having a 16-bp spacer.

Base Sequence↗
PubMed1996

Genetic and sequence analysis of the cos region of the temperate Pseudomonas aeruginosa bacteriophage, D3.

The location and structure of the cos ends of bacteriophage D3, which infects Pseudomonas aeruginosa strain PAO, has been determined using a combination of deletion analysis, transposon mutagenesis, and sequencing directly off the phage DNA. Phage D3 was found to have 9-bp 3' cos ends, making it the first phage of a Gram-negative organism known to have 3' cos ends. A 700-bp region flanking the cos site was necessary for efficient transduction of D3 cosmid derivatives. This region was found to contain incomplete inverted repeat sequences flanking the cos site, along with adenine-rich repeats homologous to coliphage gama Ter binding sites. Possible IHF binding sites were also present.

Base Sequence↗
PubMed1996

Physical mapping of several heat-shock genes in Pseudomonas aeruginosa and the cloning of the mopA (GroEL) gene.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide or amino acid motifs in heat-shock genes/proteins, we have physically mapped the dnaK, lon, and hptG genes of Pseudomonas aeruginosa. Hybridization data suggest that there is a single copy of the mopBA (GroES/GroEL) operon but several additional copies of mopA. In addition, the map coordinates for the rpoD, rpoS, and rpoH genes were determined. The mopA gene from the mopBA operon was cloned and sequenced. The protein product of this gene showed 79% amino acid identity to the Escherichia coli GroEL and 98% identity to the GroEL sequence from P. aeruginosa ATCC 27853. A number of discrepancies were found with the latter sequence.

Amino Acid Sequence↗
PubMed1995

Cloning the gene for the heat shock response positive regulator (sigma 32 homolog) from Pseudomonas aeruginosa.

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040-1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (sigma 32) of E. coli. The plasmid containing the rpoH gene complemented the function of sigma 32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription-translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 degrees C) conditions. Two promoter sites were identified having sequence homology to the E. coli sigma 70 and sigma 24 consensus sequences.

Amino Acid Sequence↗
PubMed1994

Sinusoidal 60 Hz electromagnetic fields failed to induce changes in protein synthesis in Escherichia coli.

Escherichia coli JM83 [F- ara delta(lac-proAB) rpsL [phi 80d delta (lacZ)M15]] in midlog growth phase at 30 degrees C were exposed to 60 Hz sinusoidal magnetic field of 3 mT of nonuniform diverging flux, inducing a nonuniform electric field with a maximum intensity of 32 microV/cm using an inductor coil. Exposed and unexposed control cells were maintained at 30.8 +/- 0.1 degrees C and 30.5 +/- 0.1 degrees C, respectively. Quadruplicate samples of exposed and unexposed E. coli cells were simultaneously radiolabeled with 35S-L-methionine at 10 min intervals over 2 hr. Radiochemical incorporation into proteins was analyzed via liquid scintillation counting and by denaturing 12.5% polyacrylamide gel electrophoresis. The results showed that E. coli exposed to a 60 Hz magnetic field of 3 mT exhibited no qualitative or quantitative changes in protein synthesis compared to unexposed cells. Thus small prokaryotic cells (less than 2 microns x 0.5 micron) under constant-temperature conditions do not alter their protein synthesis following exposure to 60 Hz magnetic fields at levels at 3 mT.

Autoradiography↗
PubMed1994

Nucleotide sequence of the Pseudomonas aeruginosa insertion sequence IS222: another member of the IS3 family.

Sequence analysis of the Pseudomonas aeruginosa insertion sequence element IS222 revealed it to be 1234 bp in size with 23 bp imperfect terminal inverted repeats. Insertion caused a 5-bp duplication of the insertion site. Two ORFs were identified, one of which, ORFA, could encode a basic (pI 10.5) polypeptide with a mass of 11,709. This sequence bears strong homology to the putative ORFA product from the Shigella dysenteriae insertion sequence element IS911, which is a member of the IS3 family of insertion elements. As with other members of this group the nucleotide sequence contains a "frameshift window" (AAAAAAG; M. Chandler and O. Fayet (1993). Mol. Microbiol. 7, 497-503) at which ribosome slippage can result in a fusion protein (ORFAB).

Amino Acid Sequence↗
PubMed1994

Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host-range plasmids of the pQF series.

The nucleotide sequence of the replicative origin (OriR) region of the small cryptic broad-host-range plasmid, pRO1600, which forms the basis of a number of useful cloning vectors has been determined. In addition it has been subjected to Tn5 mutagenesis, deletion analysis, and subcloning in order to define the regions essential for replication in Pseudomonas aeruginosa. The sequence (1894 bp) contains a fragment derived from transposon Tn1. The OriR region is structurally related to other replication (Rep) protein-dependent origins in that it has an A-T-rich region upstream of four 17-bp direct repeats (iterons) which presumably function in initiator protein binding. The sequence also contains a DNA-A-binding site and an open reading frame which could encode a basic (pI 10.6) 25,343-Da Rep protein with homology to RepA from the Neisseria gonorrhoeae beta-lactamase plasmid pFA3. The possible evolutionary origin of this plasmid in P. aeruginosa (RP1) is discussed.

Amino Acid Sequence↗
PubMed1994

Cloning of the early promoters of Pseudomonas aeruginosa bacteriophage D3: sequence of the immunity region of D3.

The early promoters of bacteriophage D3 of Pseudomonas aeruginosa were cloned and physically mapped to the right 25% of the phage genome. The promoters were cloned into promoter selection vector pQF26, and their relative strengths, the direction of transcription, and whether they were directly regulated by repressor were determined. A 3.3-kb fragment of the genome containing the immunity region was sequenced and analyzed (GenBank accession number: L22692). The promoter activity associated with this region was determined to be bidirectional and repressible, indicating that this region contains operator-promoter complexes. Sequence and functional analyses suggest that this region is analogous to the immunity region of coliphage lambda. Two strong promoters, one of which was repressible, were found to be located adjacent to the immunity region. Clear-plaque mutant phage D3c contains insertion element IS222, which causes it to behave as a repressor-negative (c1) variant. The site of insertion of IS222 was sequenced and determined to lie within the c1 gene open reading frame. This phage shows remarkable similarity in genomic organization to coliphage lambda and its relatives.

Amino Acid Sequence↗
PubMed1993

Localization of the virulence-associated genes pilA, pilR, rpoN, fliA, fliC, ent, and fbp on the physical map of Pseudomonas aeruginosa PAO1 by pulsed-field electrophoresis.

Seven virulence-associated genes have been placed on a genomic map of Pseudomonas aeruginosa PAO1, using pulsed-field electrophoresis, on the basis of the previous physical maps of Romling et al. (U. Romling, M. Duchene, D. Essar, D. Galloway, C. Guidi-Rontani, D. Hill, A. Lazdunski, R. Miller, K. Schleifer, D. Smith, H. Toschka, and B. Tummler, J. Bacteriol. 174:327-330, 1992; U. Romling, D. Grothues, W. Bautsch, and B. Tummler, EMBO J. 8:4081-4089, 1989) and Ratnaningsih et al. (E. Ratnaningsih, S. Dharmsthiti, V. Krishnapillai, A. Morgan, M. Sinclair, and B. W. Holloway, J. Gen. Microbiol. 136:2351-2357, 1990). The new locations for the outer membrane enterobactin iron-siderophore receptor ent gene (41 to 42 min) and the fliA gene (59 to 61 min), which encodes a minor sigma factor of RNA polymerase, are given. The pilA (the pilin structural gene), pilR (a pilin regulatory gene), and rpoN (encoding another minor sigma factor of RNA polymerase) genes map together at 71 to 75 min, locations correcting the previously reported values (V. Shortridge, M. Pato, A. Vasil, and M. Vasil, Infect. Immun. 59:3596-3603, 1990). The fbp gene (28 to 29 min), which encodes an outer membrane ferripyochelin-binding protein of low molecular weight, and the fliC gene (64 to 66 min), the flagellin structural gene, were determined to lie in the previously reported locations.

Bacterial Outer Membrane Proteins↗
PubMed1991

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.

Archaea↗
PubMed1990

Construction of broad-host-range vectors for the selection of divergent promoters.

A series of promoter-probe plasmid vectors has been constructed which allows for the selection of DNA sequences containing divergent control elements. Each vector contains a pair of promoterless genes [encoding beta-galactosidase (lacZ), alkaline phosphatase (phoA), and bacterial luciferase (luxAB)] arranged in an antiparallel fashion and separated by a large intervening multiple cloning site. The vectors permit direct detection of promoter activity on indicator plates after transformation. Cloned promoters are selected based on production of coloured products in the case of lacZ and phoA, and by the emission of light in the case of luxAB. These vectors have been tested using known divergent promoter elements from pBR322 and Pseudomonas phage D3.

Cloning, Molecular↗
PubMed1990

Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene.

Expression studies utilizing the regA promoters, fused in tandem or separately to promoterless reporter genes, indicated that regA is transcribed from two promoters (P1 and P2). Both promoters can act independently. Expression from the P1 promoter is not affected by the iron content of the medium. Expression from the P2 promoter is tightly regulated by iron.

ADP Ribose Transferases↗
PubMed1990

High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions.

High efficiency transformation of Pseudomonas aeruginosa was achieved using frozen cell suspensions and high voltage electroporation. We have obtained frequencies as high as 5.8 x 10(8) transformants/micrograms of plasmid DNA using PA01 strain OT684 and a buffer of 15% glycerol-1 mM MOPS. The method allows for easy and reproducible production of frozen cell suspensions for rapid transformation of P. aeruginosa.

DNA, Bacterial↗
PubMed1990

Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters.

We have constructed a series of broad-host-range plasmids which use "visual screens" to detect promoter activity. These plasmids contain the pMB1 and pRO1600 origins of replication and are capable of replicating in a wide range of gram-negative bacteria. The genes encoding beta-galactosidase and alkaline phosphatase from Escherichia coli and bacterial luciferase from Vibrio harveyi supply the promoterless indicator genes. The constructs were tested in E. coli and Pseudomonas aeruginosa.

Alkaline Phosphatase↗
PubMed1990

Transduction of a plasmid containing the bacteriophage D3 cos site in Pseudomonas aeruginosa.

Plasmids harboring the cos sequences of bacteriophage D3 can be transferred, by bacteriophage D3, into Pseudomonas aeruginosa by a mechanism which is insensitive to DNase. Transducing activity was separated from the plaque-forming particles by CsCl equilibrium gradient centrifugation. Restriction endonuclease digestion patterns suggest that the transducing particles contain plasmid concatemers.

Bacteriophages↗
PubMed1989

Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli.

We have constructed two promoter-selection vectors based upon the broad-host-range plasmid pRO1614. pQF40 (6 kb) contains a promoterless tetA gene downstream from a large multiple cloning site while pQF26 (5.4 kb) possesses a promoterless cat cartridge. The latter vector displayed a copy number of 13 in Pseudomonas aeruginosa and 39 in Escherichia coli. When promoter sequences derived from the Pseudomonas phage phi PLS27 were cloned into pQF26, high levels of chloramphenicol-acetyltransferase were detected in P. aeruginosa. In E. coli the activity was approximately one-third that in P. aeruginosa when corrections were made for the plasmid copy number.

Bacteriophages↗