Clustering of the trp genes in Burkholderia (formerly Pseudomonas) cepacia

Abstract Chromosomal location of trp genes of a strain Burkholderia cepacia (formerly Pseudomonas cepacia ) has been determined by transduction using a generalized transducing phage CP75 and by molecular analysis for a cosmid plasmid clone with trp genes isolated from the genomic gene library of the strain. The trp genes were classified into three linkage groups and they all were closely linked on a short chromosomal region probably in the order ( trpA, trpB, trpF)-(trpC, trpD)-trpE .     

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.     

Indirect mutagen assay in human lymphocyte in the presence of a metabolic activation system

Chromosome aberrations in cultured human lymphocytes were examined after exposures to various concentrations (from 1 X 10(-6) to 1 X 10(-3) mol X l-1) of cyclophosphamide (CP) in the presence or absence of a metabolic activation system (S9 mix). With metabolic activation, increases in the frequency of aberrant cells (AB. C.) produced by CP were significant and dose-dependent. At a concentration of 5 X 10(-4) mol X l-1, activated CP induced 29% AB. C. versus 6% AB. C. detected after exposures to CP without metabolic activation. The freshly prepared S9 mix did not virtually differ in its activation potency from the S9 mix stored for 3 weeks at -20 degrees C. CP preincubated for 100 min with S9 mix caused little or no increase in AB. C. frequency above the control level.     

Large macromolecules can be introduced into cultured mammalian cells using erythrocyte membrane vesicles

Plasmid 6.4 kbp DNA, 14 kbp DNA, lambda phage particles, all of which contained herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, or IgM molecules, were mixed with erythrocyte membranes and treated with neutral detergent. The transparent mixture was diluted with phosphate-buffered saline (PBS), followed by centrifugation to collect membrane vesicles containing the large macromolecules. 10-15% of 6.4 kbp, 3% of 14 kbp, 4-7% of the lambda phage particles and 14.5% of IgM were trapped within erythrocyte membrane vesicles. The membrane vesicles containing these molecules were fused with L cells, or rat F2408#20 cells, both of which are deficient in thymidine kinase activity. In each case, transformants were obtained. 2 X 10(5) - 7 X 10(5) phage PFU or 1.5 X 10(6) - 8 X 10(7) DNA molecules were required to obtain one transformant from L cells, but 2-3 X 10(7) phage PFU or 2 X 10(9) - 1 X 10(10) DNA molecules were required for one transformant from rat cells. Number of colonies which transiently expressed TK genes in L cells was also determined by autoradiography. The ratio of stable transformants to colonies positive for transient expression in cells treated with low doses of DNA or lambda phage was 46-68%. The transformation efficiency of human fibroblast cells by pSV2-gpt DNA trapped in erythrocyte membrane vesicles was less than that of L cells by HSV-TK DNA, but almost the same as that of rat cells by HSV-TK DNA.     

Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).     

Seasonal Population Dynamics and Interactions of Competing Bacteriophages and Their Host in the Rhizosphere

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-Φ1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as ΦCP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage ΦCP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some ΦCP6-1 phage up to day 137, yet hardly any ΦCP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.     

Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv. campestris.

We have isolated and characterized a lytic double-stranded DNA Xanthomonas campestris pv. campestris bacteriophage (XTP1) capable of mediating generalized transduction. The phage transduces chromosomal markers at frequencies of 10(-5) to 10(-6) transductants per PFU. We demonstrated its genetic utility by the isolation and cotransduction of linked transposon insertions to a nonselectable locus, xgl, required for the cleavage of 5-bromo-3-chloro-indoyl-beta-D-galactoside and showed that rif and str alleles in X. campestris are 75% linked. One-step growth experiments showed that the latent and rise periods were each 2 h and the average burst size was 35. The DNA genome is approximately 180 kb, presumably modified in a sequence-specific manner, and may be covalently attached to protein(s). Electron micrographs show the phage particle to have an icosahedral head and contractile tail with tail fibers uniquely attached to a location 40 nm proximal from the end of the tail.     

Seasonal population dynamics and interactions of competing bacteriophages and their host in the rhizosphere

We describe two prolonged bacteriophage blooms within sugar beet rhizospheres ensuing from an artificial increase in numbers of an indigenous soil bacterium. Further, we provide evidence of in situ competition between these phages. This is the first in situ demonstration of such microbial interactions in soil. To achieve this, sugar beet seeds were inoculated with Serratia liquefaciens CP6RS or its lysogen, CP6RS-ly-phi 1. These were sown, along with uninoculated seeds, in 36 field plots arranged in a randomized Latin square. The plots were then sampled regularly over 194 days, and the plants were assayed for the released bacteria and any infectious phages. Both the lysogen and nonlysogen forms of CP6RS survived equally well in situ, contradicting earlier work suggesting lysogens have a competitive disadvantage in nature. A Podoviridae phage, identified as phi CP6-4, flourished on the nonlysogen-inoculated plants in contrast to those plants inoculated with the lysogen. Conversely, the Siphoviridae phage phi CP6-1 (used to construct the released lysogen) was isolated abundantly from the lysogen-treated plants but almost never on the nonlysogen-inoculated plants. The uninoculated plants also harbored some phi CP6-1 phage up to day 137, yet hardly any phi CP6-4 phages were found, and this was consistent with previous years. We show that the different temporal and spatial distributions of these two physiologically distinct phages can be explained by application of optimal foraging theory to phage ecology. This is the first time that such in situ evidence has been provided in support of this theoretical model.     

Transduction ability of mutants of phage CP51, virulent for bacteria of the Bacillus cereus group

The virulent phage CP51 used usually to transfer chromosomal and plasmid markers between bacteria of the Bacillus cereus group was treated with N-methyl-N'-nitro-N-nitroguanidine. Mutants with reduced viability and ts-mutants were isolated. Some of the mutants were found to have an increased efficiency of transduction and allow to simplify the process. Transfer frequencies of the plasmid pBC16 by the phages CP51-26 and CP51-4-59 were 5 x 10(-4) per plaque-forming unit and 4-5 x 10(-3) per bacterial cell, respectively. Possibilities of further increasing the transduction efficiency of Bacillus thuringiensis genetic material using phage CP51 mutants are discussed.     

Resistance plasmids in Pseudomonas cepacia 4G9.

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).     

In Situ Population Dynamics of Bacterial Viruses in a Terrestrial Environment

Predation by bacteriophages is thought to control bacterial numbers and facilitate gene transfer among bacteria in the biosphere. A thorough understanding of phage population dynamics is therefore necessary if their significance in natural environments is to be fully appreciated. Here we describe the in situ population dynamics of three separate phage populations predating on separate bacterial species, living on the surface of field-grown sugar beet (Beta vulgaris var. Amethyst), as recorded over a 9-month period. The distributions of the three phage populations were different and fluctuated temporally in 1996 (peak density, ~10³ PFU g⁻¹). One of these populations, predating on the indigenous phytosphere bacterium Serratia liquefaciens CP6, consisted of six genetically distinct DNA phages that varied in relative abundance to the extent that an apparent temporal succession was observed between the two most abundant phages, ΦCP6-1 and ΦCP6-4.     

Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library

The substrate specificity of human collagenase 3 (MMP-13), a member of the matrix metalloproteinase family, is investigated using a phage-displayed random hexapeptide library containing 2 x 10(8) independent recombinants. A total of 35 phage clones that express a peptide sequence that can be hydrolyzed by the recombinant catalytic domain of human collagenase 3 are identified. The translated DNA sequence of these clones reveals highly conserved putative P1, P2, P3 and P1', P2', and P3' subsites of the peptide substrates. Kinetic analysis of synthetic peptide substrates made from human collagenase 3 selected phage clones reveals that some of the substrates are highly active and selective. The most active substrate, 2, 4-dinitrophenyl-GPLGMRGL-NH(2) (CP), has a k(cat)/K(m) value of 4.22 x 10(6) m(-)(1) s(-)(1) for hydrolysis by collagenase 3. CP was synthesized as a consensus sequence deduced from the preferred subsites of the aligned 35 phage clones. Peptide substrate CP is 1300-, 11-, and 820-fold selective for human collagenase 3 over the MMPs stromelysin-1, gelatinase B, and collagenase 1, respectively. In addition, cleavage of CP is 37-fold faster than peptide NF derived from the major MMP-processing site in aggrecan. Phage display screening also selected five substrate sequences that share sequence homology with a major MMP cleavage sequence in aggrecan and seven substrate sequences that share sequence homology with the primary collagenase cleavage site of human type II collagen. In addition, putative cleavage sites similar to the consensus sequence are found in human type IV collagen. These findings support previous observations that human collagenase 3 can degrade aggrecan, type II and type IV collagens.     

Inoculum size as a factor limiting success of inoculation for biodegradation

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Inoculum size as a factor limiting success of inoculation for biodegradation.

A study was conducted to determine the role of inoculum size of a bacterium introduced into nonsterile lake water in the biodegradation of a synthetic chemical. The test species was a strain of Pseudomonas cepacia able to grow on and mineralize 10 ng to 30 micrograms of p-nitrophenol (PNP) per ml in salts solution. When introduced into water from Beebe Lake at densities of 330 cells per ml, P. cepacia did not mineralize 1.0 microgram of PNP per ml. However, PNP was mineralized in lake water inoculated with 3.3 X 10(4) to 3.6 X 10(5) P. cepacia cells per ml. In lake water containing 1.0 microgram of PNP per ml, a P. cepacia population of 230 or 120 cells per ml declined until no cells were detectable at 13 h, but when the initial density was 4.3 X 10(4) cells per ml, sufficient survivors remained after the initial decline to multiply at the expense of PNP. The decline in bacterial abundance coincided with multiplication of protozoa. Cycloheximide and nystatin killed the protozoa and allowed the bacterium to multiply and mineralize 1.0 microgram of PNP, even when the initial P. cepacia density was 230 or 360 cells per ml. The lake water contained few lytic bacteria. The addition of KH2PO4 or NH4NO3 permitted biodegradation of PNP at low cell densities of P. cepacia. We suggest that a species able to degrade a synthetic chemical in culture may fail to bring about the same transformation in natural waters, because small populations added as inocula may be eliminated by protozoan grazing or may fail to survive because of nutrient deficiencies.     

Selective medium for Pseudomonas cepacia containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate.

Contamination of solutions and lotions with Pseudomonas cepacia is a growing concern among health professionals. The identification of P. cepacia usually requires a long series of biochemical tests. In an effort to develop a more direct method, we evaluated plate count agar containing 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate at respective concentrations of 1 and 75 micrograms/ml as a medium for selectively isolating P. cepacia. The medium inhibited the growth of all gram-negative bacilli and gram-positive cocci tested except P. cepacia and Serratia marcescens. These two microorganisms could easily be differentiated by their colony morphology and their reactions in the oxidase test. When nonsterilized water samples were inoculated with P. cepacia and spread or streaked on the selective medium, all P. cepacia organisms were recovered. These results demonstrate the usefulness of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan and polymyxin B sulfate in the detection of P. cepacia. We believe that this selective medium could be useful in isolating P. cepacia from mixed bacterial flora that might be present in environmental water and water-related samples, such as solutions and lotions.     

Mutagenesis of free and intracellular cyanophage AS-1 by ultraviolet, N-methanyl-N'-nitro-N-nitrosoguanidine and acriflavine.

Mutagenic actions of ultraviolet irradiation (UV), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and acriflavine (photodynamic) were tested in free and intracellular phage AS-1 infecting Anacystis nidulans IU625. Spontaneous and induced mutations with particular reference to host range (h) and minute plaque formation (m) were investigated. The spontaneous mutation frequencies varied from 10(-9) to 10(-8) and from 2 X 10(-5) to 2 X 10(-4) for h and m mutants respectively. UV was efficient in inducing h and m markers in free phage particles as well as intracellular phage; MNNG induced both markers in intracellular phage only, and acriflavine induced m mutants only in free as well as in infecting phages. UV-induced mutations in free phage were photo-reactivable by visible light. With all the mutagens used, maximal induction of both markers was observed with treatment of 2-h complexes.     

Frequency of infection and efficiency of transfection of Rhizobium meliloti cells and spheroplasts.

Competent cultures of Rhizobium meliloti cells and spheroplasts obtained by various methods were infected with DNA of phage 1A. The Frequency of infection among the cells and spheroplasts was 2 X 10(-8)-5 X 10(-10). The efficiency of transfection calculated from the ratio of plaque forming units to infective DNA molecule of phage 1A was 5 X 10(-8) to 10(-10). Frequency of infection and efficiency of transfection among the competent cells were by one order of magnitude higher in the case of the spheroplasts. The use of various media did not noticeably alter the efficiency of transfection.     

Structure of Marek's disease virus DNA: detailed restriction enzyme map.

Purified virion DNA (120 X 10(6) molecular weight [MW]) of Marek's disease virus strain GA was cleaved with BamHI restriction endonuclease, and 27 out of the 29 fragments were cloned into bacterial plasmids. Restriction maps for BamHI, BglI, and SmaI endonucleases were constructed. The genomic structure of Marek's disease virus DNA was found to be similar to that of herpes simplex virus types 1 and 2. A long unique region (75 X 10(6) MW, located at 10 X 10(6) to 85 X 10(6) MW [10-85] from the left end of the genome), which was subdivided into segment 1 (22 X 10(6) MW, located at 10-32) and segment 2 (51 X 10(6) MW, located at 34-85) by direct repeats (32-34), was flanked by a long terminal region (10 X 10(6) MW, located at 0-10) and a long inverted region (10 X 10(6) MW, located at 85-95). A short unique region (8 X 10(6) MW, located at 103-111) was flanked by a short terminal region (8 X 10(6) MW, located at 111-119) and a short inverted region (8 X 10(6) MW, located at 95-103). The direct repeat fragments (0.9 X 10(6) could be isolated by cleavage with SmaI. The right terminal end was found to be heterogenous .     

Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage.

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.     

Transduction of Pseudomonas aeruginosa with a mutant of bacteriophage E79.

A mutant of the virulent bacteriophage E79 was isolated which mediated generalized transduction in Pseudomonas aeruginosa. Variable recovery of transductants as a result of phage killing was avoided by the use of recipients carrying the IncP-2 plasmid R38, and transduction frequencies of 4 X 10(-6) to 1 X 10(-5) per plaque-forming unit were obtained. Linkage studies have indicated that the coinheritance frequencies are less than would be expected from the published molecular weight of E79 deoxyribonucleic acid (120 X 10(6). By using recipients carrying R38, low-frequency transduction by wild-type E79 and two other virulent phages, F8 and phi 16, was demonstrated.