Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny.

Mutants of Pseudomonas aeruginosa with impaired ability to establish a lysogenic relationship with temperate bacteriophage (Les-) have been isolated. These les mutations map to two areas of the P. aeruginosa chromosomal map as determined by conjugational and transductional analyses. Two phenotypic classes of Les- mutants were identified. One class of mutations has pleiotropic effects on DNA metabolism. These mutants are unable to recombine genetic material acquired as a result of either conjugation or transduction (Rec-). In addition, the ability of these Les- Rec- mutants to repair UV-induced damage to bacteriophage is reduced (host-cell reactivation deficient, Hcr-). Mutants of the second class are Les-, Rec+, and Hcr+.     

x811--mutation of the Pseudomonas aeruginosa transposable phage D3112 with a pleiotropic effect]

Characteristics of Pseudomonas aeruginosa Transposable Phage D3112 carrying mutation x811 are described. x811 is a recessive mutation with pleiotropic effect. It determines a deteriorated lysis of infected or induced bacteria, a delayed replication, and a considerably decreased replication rate. In addition, the x811 mutation is expressed as the Kil phenotype, since high-temperature induction of prophage D3112 cts15 x811 does not cause an immediate decrease in the ability of bacteria to form colonies at 42 degrees C. Restriction analysis of DNA of D3112 cts15 x811 and its segregants has not revealed extended insertions or deletions. The characteristics of segregants of the D3112 cts15 x811 phage agree with the suggestion that the x811 mutation has emerged in a regulatory element (a gene or a site) that controls both expression of the entire early operon, including the "pre-early" function Kil, and the regulation of the repressor synthesis.     

Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site

Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization.     

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.     

Intestinal Tissues Induce an SNP Mutation in Pseudomonas aeruginosa That Enhances Its Virulence: Possible Role in Anastomotic Leak

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (～10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1- termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak.     

Cloning and characterization of the c1 repressor of Pseudomonas aeruginosa bacteriophage D3: a functional analog of phage lambda cI protein.

We cloned the gene (c1) which encodes the repressor of vegetative function of Pseudomonas aeruginosa bacteriophage D3. The cloned gene was shown to inhibit plating of D3 and the induction of D3 lysogens by UV irradiation. The efficiency of plating and prophage induction of the heteroimmune P. aeruginosa phage F116L were not affected by the presence of the cloned c1 gene of D3. When the D3 DNA fragment containing c1 was subcloned into pBR322 and introduced into Escherichia coli, it was shown to specifically inhibit the plating of phage lambda and the induction of the lambda prophage by mitomycin C. The plating of lambda imm434 phage was not affected. Analysis in minicells indicated that these effects correspond to the presence of a plasmid-encoded protein of 36,000 molecular weight. These data suggest the possibility that coliphage lambda and the P. aeruginosa phage D3 evolved from a common ancestor. The conservation of the functional similarities of their repressors may have occurred because of the advantage to these temperate phages of capitalizing on the potential of the evolutionarily conserved RecA protein to monitor the level of damage to the host genome.     

Effect of bacteriophage C5 on ultraviolet light survival in Pseudomonas aeruginosa.

Bacteriophage C5 of Pseudomonas aeruginosa is able to reactivate ultraviolet (u.v.)-irradiated phage E79 in coinfection experiments and decrease the u.v.-sensitivity of a host-cell reactivation deficient mutant. These properties suggest that phage C5 has a gene(s) which is involved in the repair of u.v.-damaged DNA. The isolation of two u.v.-sensitive mutants of C5 supports this hypothesis.     

Pleiotropic effects of the mioC mutation on the physiology of Pseudomonas aeruginosa PAO1

Flavodoxin (Fld) is a bacterial electron-transfer protein that possesses flavin mononucleotide as a prosthetic group. In the genomes of the Pseudomonas species, the mioC gene is the sole gene, annotated Fld, but its function remains unclear. In this study, phenotype microarray analysis was performed using the wild-type and mioC mutant of pathogenic Pseudomonas aeruginosa PAO1. Our results showed that the mioC mutant is very resistant to oxidative stress. Different antibiotics and metals worked differently on the sensitivity of the mutant. Other pleiotropic effects of mutation in the mioC gene, such as biofilm formation, aggregation ability, motility and colony morphology, were observed under iron stress conditions. Most of the phenotypic and physiological changes could be recovered in the wild type by complementation. Mutation of the mioC gene also influenced the production of pigments. The mioC mutant and mioC over-expressed complementation cells, over-produced pyocyanin and pyoverdine, respectively. Various secreted chemicals were also changed in the mutant, which was confirmed by (1) H NMR analysis. Interestingly, physiological alterations of the mutant strain were restored by the cell-free supernatant of the wild type. The present study demonstrates that the mioC gene plays an important role in the physiology of P.?aeruginosa and might be considered as a suitable drug target candidate in pathogenic P.?aeruginosa.     

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.     

Construction of plasmids that produce phage P22 repressor

In a series of plasmid constructions, the c2 (repressor) gene of phage P22 was cloned in a multicopy plasmid and expressed at increasing level. The final result of these constructions is a plasmid that maintains a level of approx. 200 times as much repressor as is found in a lysogen. A series of increasingly virulent phage mutants was isolated by plating sequentially on host cells with increasing levels of repressor. The methods used in the constructions should be applicable to obtaining elevated expression of cloned genes in other systems.     

A new pleiotropic bacteriophage P1 mutation, bof, affecting c1 repression activity, the expression of plasmid incompatibility and the expression of certain constitutive prophage genes.

Summary In bacteriophage P1 an amber mutation in a new gene, bof, has been isolated. The bof-1 phage mutant exhibits a pleiotropic phenotype; bof product is non-essential, and acts as a positive modulator. In P1 bac-1 mutants, in which a dnaB analog product, ban, is expressed constitutively, the bof product activates ban expression both in the prophage state and in lytic growth: P1 bof bac prophages have a reduced ban activity and in lytic growth P1 bof bac phages show a lower ban activity than P1 wild type. This effect on ban activity is observed specifically in P1 bac-1 mutants; it is not mediated by the cl repressor of the lytic functions (repressor of the ban operon) since this effect occurs even if the phage carries a heat sensitive c1 repressor. Thus we concluded that the bac mutation put the ban operon under an abnormal, unknown control, modulated by the bof product. P1 bof lysogens show an increased immunity to superinfecting P1 phage and are affected in their inducibility properties; in the presence of the altered c1-100 repressor, bof product is required for maintenance of lysogeny, as shown by the induction of P1 c1-100 bof-1 lysogens at 30°. P1 bof superinfecting phage can be established together with a resident P1 bof prophage in a recA host, unlike P1 wild type which cannot form double lysogens. P1 bof double lysogens are unstable and segregate one or the other prophage. P1 Cm bof and P1 Km bof lysogens show higher levels of antibiotic resistance than the corresponding bof ⁺ lysogens. The bof gene has been mapped, in an interval defined by P1 prophage deletion end points, far from both ban and c1. All bof phenotypes are reversed by single mutations.     

Bacteriophage T4 self-assembly: in vitro reconstitution of recombinant gp2 into infectious phage

T4 gene 2 mutants have a pleiotropic phenotype: degradation of injected phage DNA by exonuclease V (ExoV) in the recBCD(+) host cell cytoplasm and a low burst size due, at least in part, to a decreased ability for head-to-tail (H-T) joining. The more N terminal the mutation, the more pronounced is the H-T joining defect. We have overexpressed and purified the recombinant gene 2 product (rgp2) to homogeneity in order to test its role in H-T joining, during in vitro reconstitution. When we mix extracts of heads from a gp2(+) phage infection (H(+)) with tails from a gp2(+) or gp2(-) phage infection (T(+) or T(-)), the H-T joining is fast and all of the reconstituted phage grow equally well on cells with or without ExoV activity. When heads from gene 2 amber mutants (H(-)) are used, addition of rgp2 is required for H-T joining. In this case, H-T joining is slow and only about 10% of the reconstituted phage can form plaques on ExoV(+) cells. When extracts of heads with different gene 2 amber mutations are mixed with extracts of tails (with a gene 2 amber mutation) in the presence of rgp2, we find that the size of the gp2 amber peptide of the head extract is inversely related to the fraction of reconstituted phage with a 2(+) phenotype. We conclude that free rgp2 is biologically active and has a direct role in H-T joining but that the process is different from H-T joining promoted by natural gp2 that is incorporated into the head in vivo. Furthermore, it seems that gp2 has a domain which binds it to the head. Thus, the presence of the longer gp2am mutants (with this domain) inhibits their replacement by full-length rgp2.     

Escherichia coli mutants deficient in deoxyuridine triphosphatase.

Mutants deficient in deoxyuridine triphosphatase (dUTPase) were identified by enzyme assays of randomly chosen heavily mutagenized clones. Five mutants of independent origin were obtained. One mutant produced a thermolabile enzyme, and it was presumed to have a mutation in the structural gene for dUTPase, designated dut. The most deficient mutant had the following associated phenotypes: less than 1% of parental dUTPase activity, prolonged generation time, increased sensitivity to 5'-fluorodeoxyuridine, increased rate of spontaneous mutation, increased rate of recombination (hyper-Rec), an inhibition of growth in the presence of 2 mM uracil, and a decreased ability to support the growth of phage P1 (but not T4 or lambda). This mutation also appeared to be incompatible with pyrE mutations. A revertant selected by its faster growth had regained dUTPase activity and lost its hyper-Rec phenotype. Many of the properties of the dut mutants are compatible with their presumed increased incorporation of uracil into DNA and the subsequent transient breakage of the DNA by excision repair.     

Involvement of the product of the glnF gene in the autogenous regulation of glutamine synthetase formation in Klebsiella aerogenes.

Mutations in a site, glnF, linked by P1-mediated transduction of argG on the chromosome of Klebsiella aerogenes, result in a requirement for glutamine. Mutants in this gene have in all media a level of glutamine synthetase (GS) corresponding to the level found in the wild-type strain grown in the medium producing the strongest repression of GS. The adenylylation and deadenylylation of GS in glnF mutants is normal. The glutamine requirement of glnF mutants could be suppressed by mutations in the structural gene for GS, glnA. These mutations result in altered regulation of GS synthesis, regardless of the presence or absence of the glnF mutation (GlnR phenotype). In GlnR mutants the GS level is higher than in the wild-type strain when the cells are cultured in strongly repressing medium, but lower than in the wild-type strain when cells are cultured in a derepressing medium. Heterozygous merodiploids carrying a normal glnA gene as well as a glnA gene responsible for the GlnR phenotype behave in every respect like merodiploids carrying two normal glnA genes. These results confirm autogenous regulation of GS synthesis and indicate that GS is both a repressor and an activator of GS synthesis. The mutation in glnA responsible for the GLnR phenotype has apparently resulted in the formation of a GS that is incompetent both as repressor and as activator of GS synthesis. According to this hypothesis, the product of the glnF gene is necessary for activation of the glnA gene by GS.     

Analysis of forward mutations induced by N-methyl-N''-nitro-N-nitrosoguanidine in the bacteriophage P22 mnt repressor gene.

We describe the isolation and genetic characterization of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations in the phage P22 mnt repressor gene cloned in plasmid pBR322. Mutations in the mnt repressor gene or its operator on this plasmid, pPY98, confer a tetracycline resistance phenotype, whereas the wild-type plasmid confers tetracycline sensitivity. Cells carrying pPY98 were briefly exposed to MNNG to give 20 to 40% survival and a 50- to 100-fold increase in tetracycline-resistant cells. DNA sequence analysis showed that 29 of 30 MNNG-induced mutations were GC-to-AT transitions and one was an AT-to-GC transition. About 80% of the mutations are in three hotspots. This mutation spectrum is consistent with the proposed mechanism of mutagenic action of MNNG, which involves mispairing of an alkylated base, O6-methylguanine. The mnt gene may be a useful target for determining mutagenic specificity at the nucleotide level because forward mutations are easily isolated, the target size is small, and the DNA sequence changes of mutations can be determined rapidly.     

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.     

Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.