Biomedical subjects

V Deretic

Publications and source records attributed to V Deretic.

At least 73 records · Page 4Linked to original sources

PubMed1993

Differentiation of Pseudomonas aeruginosa into the alginate-producing form: inactivation of mucB causes conversion to mucoidy.

Mucoidy in Pseudomonas aeruginosa is a critical virulence factor associated with chronic respiratory infections in cystic fibrosis. A cluster of three tightly linked genes, algU, mucA and mucB located at 67.5 min, controls development of mucoid phenotype. This locus is allelic with a group of mutations (muc) associated with conversion into constitutively mucoid forms. One of the genes previously characterized in this region, algU, is absolutely required for the transcriptional activation of algD, a critical event in the establishment of mucoidy. AlgU is homologous to the alternative sigma factor sigma H (Spo0H) controlling sporulation and competence in Bacillus. Two genes downstream of algU, mucA and mucB were further characterized in this study. Previous complementation studies have demonstrated that mucA is required for suppression of mucoidy in the muc-2 strain PAO568. In this work, complementation analysis indicated that, in addition, mucB was required for suppression of mucoidy in the muc-25 strain PAO581, and for enhanced complementation of the muc-2 mutation in PAO568. The complete nucleotide sequence of mucA and mucB was determined. Insertional inactivation of mucB on the chromosome of the standard genetic strain PAO resulted in mucoid phenotype, and in a strong transcriptional activation of algD. Thus, a loss of mucB function is sufficient to cause conversion of P. aeruginosa into the mucoid phenotype. Since the algU-mucA-mucB region is a general site where muc mutations have been mapped, it is likely that mucB participates in the emergence of mucoid forms. Both mucA and mucB play a regulatory role in concert with the sigma-like factor AlgU; all three genes, along with signal transduction and histone-like elements, control differentiation of P. aeruginosa into the mucoid phenotype.

Alginates↗

PubMed1993

Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor.

Overproduction of the exopolysaccharide alginate by Pseudomonas aeruginosa results in mucoid colony morphology and is an important virulence determinant expressed by this organism in cystic fibrosis. Mucoidy is transcriptionally regulated by signal transduction systems and histone-like elements. One point of convergence of regulatory elements controlling mucoidy is the algD promoter. A newly described genetic locus required for algD transcription was characterized in this study. This DNA region, cloned from a nonmucoid PAO strain, was initially isolated on the basis of its ability to suppress mucoidy when present on a plasmid. The suppressing activity was observed in several mucoid PAO derivatives, including strain PAO568, in which the mapped muc-2 mutation is responsible for its mucoid phenotype, and in close to 40% of cystic fibrosis strains tested. Protein expression studies detected two polypeptides with apparent molecular masses of 27.5 and 20 kDa encoded by the region required for the suppression activity. The gene encoding the polypeptide with an apparent molecular mass of 27.5 kDa, termed algU, was further characterized. A functional chromosomal copy of algU was found to be necessary for the expression of mucoidy. Insertional inactivation of algU on the chromosome of the mucoid strain PAO568 abrogated alginate production and algD transcription. DNA sequence analysis revealed sequence similarity of the predicted algU gene product with sigma H (Spo0H), a sigma factor involved in the control of sporulation and competence in Bacillus spp. Physical mapping revealed that algU resided on the same SpeI fragment (F) as did the pruAB locus, known to be tightly linked with genetic determinants (muc) which can confer mucoidy in genetic crosses. When the chromosomal algU copy was tagged with a Tcr cassette (algU::Tcr), a tight genetic linkage of algU with pruAB was demonstrated by F116L-mediated generalized transduction. Moreover, algU::Tcr derivatives of PAO568 (originally carrying the muc-2 marker) lost the ability to transfer mucoidy in genetic crosses. These results suggest that algU, a regulator of algD transcription showing sequence similarity to an alternative sigma factor, and the genes immediately downstream of algU may be associated with a locus participating in the differentiation into the mucoid phenotype.

Alginates↗

PubMed1993

The algD promoter: regulation of alginate production by Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa mutants that overproduce the exopolysaccharide alginate and assume mucoid phenotype are associated with the establishment of chronic respiratory disease in cystic fibrosis. The initially invading strains are nonmucoid and frequently convert into the mucoid form. Mucoidy is regulated at the transcriptional level, mainly at the promoter of the algD gene. Control of the algD promoter represents a cooperative effort of several types of regulatory elements including bacterial signal transduction factors (principally through the response regulator AlgR) and histone like elements (e.g., Hp1 and possibly IHF). Our more recent studies have shown that conversion to mucoidy is a result of mutations in the muc genes within the algU-mucA-mucB cluster. The algU gene encodes a protein that resembles Spo0H, a sigma factor from Bacillus subtilis, which controls development of sporulation and competence. The mucA and mucB genes appear to control the activity of AlgU. Frameshift mutations that inactivate these proteins result in a strong transcriptional activation of algD, and conversion to mucoidy in both laboratory and clinical strains of P. aeruginosa.

Alginates↗

PubMed1992

In vitro interactions of the histone-like protein IHF with the algD promoter, a critical site for control of mucoidy in Pseudomonas aeruginosa.

The ability of the histone-like element Integration Host Factor (IHF) to interact with the algD promoter was investigated. IHF from Escherichia coli was found to bind to the algD promoter and to form multiple protein-DNA complexes in gel mobility shift DNA binding assay. The highest affinity binding site for IHF was mapped by DNaseI footprinting analysis. This site spanned nucleotides -50 to -85 relative to the algD mRNA start site and overlapped a sequence matching the IHF consensus sequence WATCAANNNNTTR in 12 out of 13 base pairs. Previous studies have shown that deletion of sequences including a portion of this site adversely affects algD promoter activity. IHF binding to the algD promoter induced DNA bending. Western blot analysis with antibodies against E. coli IHF detected a cross-reactive protein of a similar molecular mass in Pseudomonas aeruginosa, suggesting the presence of an analogous factor in this organism.

Amino Acid Sequence↗

PubMed1992

In vitro phosphorylation of AlgR, a regulator of mucoidy in Pseudomonas aeruginosa, by a histidine protein kinase and effects of small phospho-donor molecules.

AlgR is a transcriptional regulator of mucoidy in Pseudomonas aeruginosa, a critical virulence factor expressed in cystic fibrosis. AlgR belongs to the superfamily of bacterial signal transduction systems, and has been shown to bind to the algD promoter, a critical point in the regulation of mucoidy. This protein, like other typical response regulators, contains highly conserved residues known to be critical for the phosphorylation and signal transduction processes. However, a typical second component interacting with AlgR has not been identified. Here we demonstrate that AlgR undergoes phosphorylation in vitro when interacting with the well-characterized histidine protein kinase CheA. These results indicate that AlgR is capable of undergoing phosphorylation typical of other two-component signal transduction systems. Moreover, the phosphotransfer reaction between CheA and AlgR was found to be affected by the presence of carbamoyl phosphate, acetyl phosphate, and salts of phosphoramidic acid, recently shown to act as small-molecular-weight phospho-donors in the process of phosphorylation of several response regulators. These findings suggest that AlgR may react with intermediary metabolites such as carbamoyl phosphate and acetyl phosphate, and that these processes may play a role in the control of mucoidy in P. aeruginosa.

Amides↗

PubMed1992

AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA.

Activation of algD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely abolish inducibility of algD in response to environmental signals. In this work, a third binding site for AlgR, termed RB3, near the algD mRNA start site was characterized. Deletion of RB3 abrogated both the AlgR-binding ability and the residual inducibility of the algD promoter. DNase I footprinting analysis of RB3 resulted in a protection pattern spanning nucleotides -50 to -30. Eight of 10 residues encompassing a continuous region of protection within RB3 (positions -45 to -36) matched in the inverted orientation the conserved core sequence (ACCGTTCGTC) of RB1 and RB2. Quantitative binding measurements of AlgR association with RB1, RB2, and RB3 indicated that AlgR had significantly lower affinity for RB3 than for RB1 and RB2, with differences in the free energy of binding of 1.05 and 0.93 kcal/mol (4.39 and 3.89 kJ/mmol), respectively. Altering the core of RB2 to match the core of RB3 significantly reduced AlgR binding. Conversely, changing the core of RB3 to perfectly match the core of RB2 (mutant site termed RB3*) improved AlgR binding, approximating the affinity of RB2. RB3*, in the absence of the far upstream sites, showed an increase in activity, approaching the levels observed with the full-size algD promoter. Changing 4 nucleotides in two different combinations within the core of RB3 abolished the binding of AlgR to this site and resulted in a significant reduction of promoter activity in the presence of the far upstream sites. Thus, (i) the core sequence is essential for AlgR binding; (ii) the three binding sites, RB1, RB2, and RB3, are organized as an uneven palindrome with symmetrical sequences separated by 341 and 417 bp; and (iii) all three sites participate in algD activation.

Bacterial Proteins↗

PubMed1992

Immunocytochemical analysis of AlgP (Hp1), a histonelike element participating in control of mucoidy in Pseudomonas aeruginosa.

AlgP, a protein with an unusual carboxy-terminal domain resembling the tails of eukaryotic H1 histones, was detected in whole-cell extracts and within the cells of Pseudomonas aeruginosa by using immunoblotting and immunoelectron microscopy analyses. One known function of AlgP is its participation in the transcriptional activation of the algD gene. This is a pivotal step in the establishment of mucoidy in P. aeruginosa; mucoidy is a critical virulence factor expressed during respiratory infections in patients with cystic fibrosis. Polyclonal and monoclonal antibodies were raised against a synthetic 50-mer peptide containing two sets of six tandem repeats of the motif Lys-Pro-Ala-Ala (and its single-amino-acid substitution variants), based on the sequence of the algP gene from the standard genetic strain PAO. Western immunoblots with these antibodies and total protein extracts from P. aeruginosa revealed two polypeptides that reacted with the antibodies in all of the P. aeruginosa strains tested. The detected polypeptides displayed strain-dependent variability in their electrophoretic mobility, in accordance with the previously noted variability of the algP repeats at the DNA level. In strain PAO, the recognized polypeptides had apparent masses of 46.4 and 41.6 kDa. Immunoelectron microscopy revealed that AlgP is an intracellular protein with a wide distribution suggestive of its more general role. To indicate that fact, AlgP is given here an alternative name, Hp1. Since AlgP (Hp1) is a eubacterial histonelike element displaying sequence and domanial similarity with eukaryotic H1 histones, these findings may have implications on the understanding of the organization of the prokaryotic nucleoid and its role in the control of gene expression and bacterial virulence.

Amino Acid Sequence↗

PubMed1991

Mucoid Pseudomonas aeruginosa in cystic fibrosis: signal transduction and histone-like elements in the regulation of bacterial virulence.

The profuse production of the exopolysaccharide alginate results in mucoidy, a critical virulence factor expressed by Pseudomonas aeruginosa during chronic respiratory tract infections in cystic fibrosis. Studies of the regulation of this pathogenic determinant have unravelled at least two levels of control, including bacterial signal transduction systems and histone-like elements. Although only in its initial phase, an understanding of the dual control of mucoidy may help to illuminate adaptive processes that depend on the combination of these regulatory factors. Integration of specific signals transduced by the two-component systems with inputs generated by the general state of bacterial nucleoids may govern the expression of certain virulence determinants and provide a framework facilitating selection of phenotypes successful under particular environmental conditions and selective pressures.

Alginates↗

PubMed1991

AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site.

Strong transcriptional activation of algD, a key event in the overproduction of alginate and establishment of mucoidy in Pseudomonas aeruginosa, depends on the functional algR gene. The predicted gene product of algR shows homologies to response regulators from bacterial signal transduction systems. The algR gene was overexpressed in Escherichia coli, its product (AlgR) was purified by utilizing its apparent affinity for heparin, and its sequence was verified by partial amino acid sequence analysis. AlgR was found to interact directly with the algD promoter. Deletion mapping analysis, in conjunction with mobility shift DNA-binding assays, indicated the presence of three regions within the algD promoter capable of specifically binding AlgR. A relatively weak interaction was observed with the algD promoter fragment containing the region immediately upstream of the algD mRNA start site (-144 to +11). However, when fragments spanning regions located very far upstream from the algD mRNA initiation site (-533 and -332) were used, strong specific binding was observed. These regions were separated by a DNA segment not binding AlgR and spanning positions -332 to -144. DNase I footprinting analysis further established the presence of discrete AlgR binding sites overlapping with FUS, the far-upstream sites required for full induction of algD transcription and its environmental modulation. There were two distinct binding sites: RB1, spanning nucleotides -479 to -457, and RB2, spanning nucleotides -400 to -380. Both of these sequences shared a highly conserved core region, ACCGTTCGTC. These results established a direct interaction of AlgR with the algD promoter and revealed an arrangement of binding sites highly unusual for response regulators of the AlgR type.

Alginates↗

PubMed1990

Expression patterns of genes encoding elastase and controlling mucoidy: co-ordinate regulation of two virulence factors in Pseudomonas aeruginosa isolates from cystic fibrosis.

Transcriptional patterns of lasB and algD were compared in isogenic mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients. The lasB gene encodes elastase, a major proteolytic enzyme secreted by P. aeruginosa, while algD is required for the synthesis of alginate, an exopolysaccharide frequently overproduced by strains infecting cystic fibrosis patients. A possible coregulation at the transcriptional level of these major virulence determinants was analysed. The lasB and algD genes showed inverse levels of promoter activity. The lasB promoter was active in non-mucoid cells and inactive in mucoid cells (in four out of five tested pairs), while the algD promoter was active in mucoid cells and silent in non-mucoid cells in all cases. When PAO568, a model strain for the analysis of control of the alginate system, was grown under conditions promoting mucoidy, the algD promoter was activated, whereas lasB mRNA could not be detected. This effect was reversed when the cells were grown in a medium suppressing mucoidy. Insertional inactivation of algR, a member of the signal-transduction systems regulating algD transcription, although abolishing algD expression and rendering cells non-mucoid, did not alter the nature of the induction and repression patterns of lasB seen in the parental strain PAO568. These results suggest that the lasB gene and the alginate system are co-ordinately regulated at a level parallel to or above the algR gene.

Base Sequence↗

PubMed1990

Mucoid Pseudomonas aeruginosa in cystic fibrosis: mutations in the muc loci affect transcription of the algR and algD genes in response to environmental stimuli.

Increased levels of alginate biosynthesis cause mucoidy in Pseudomonas aeruginosa, a virulence factor of particular importance in cystic fibrosis. The algR gene product, which controls transcription of a key alginate biosynthetic gene, algD, is homologous to the activator members of the two-component, environmentally responsive systems (NtrC, OmpR, PhoB, ArcA, etc). In this report, we show that mutations in the muc loci, (muc-2, muc-22, and muc-23, in the standard genetic P. aeruginosa strain PAO, as well as a mapped muc allele in an isolate from a cystic fibrosis patient) affect transcription of algD and algR. This influence was strongly dependent on environmental factors. Regulation by nitrogen was observed in all strains examined, but the absolute transcriptional levels, determining the mucoid or nonmucoid status, were strain (muc allele)-dependent. Increased concentrations of NaCl in the medium, an osmolyte which is elevated in cystic fibrosis lung secretions, resulted in an increased algD transcription and mucoid phenotype in a muc-2 strain; the same conditions, however, produced a nonmucoid phenotype in the muc-23 background and abolished algD transcription. Mutations in the muc loci may cause mucoidy by deregulating the normal response of the alginate system to environmental stimuli.

Alginates↗

PubMed1990

A procaryotic regulatory factor with a histone H1-like carboxy-terminal domain: clonal variation of repeats within algP, a gene involved in regulation of mucoidy in Pseudomonas aeruginosa.

A novel procaryotic transcriptional regulatory element, AlgP, with a histone H1-like carboxy-terminal domain was identified in Pseudomonas aeruginosa. AlgP is required for transcription of the key biosynthetic gene algD, which is necessary for production of the exopolysaccharide alginate causing mucoidy in P. aeruginosa. Mucoidy is a critical virulence determinant of P. aeruginosa invariably associated with the respiratory infections causing high mortality in cystic fibrosis. Here we show that AlgP and histones H1 both have repeated units of the Lys-Pro-Ala-Ala motif (KPAA) and its variations within their long (over 100 amino acids) carboxy-terminal domains. This region of histone H1 tails has been shown to bind to the linker DNA in eucaryotic chromatin fibers. A synthetic 50-mer peptide consisting of repeats from the AlgP carboxy-terminal domain was found to bind DNA in a mobility shift DNA-binding assay. AlgP is encoded by a gene that contains multiple direct repeats organized as tandem, head-to-tail, 12-base-pair (bp) units overlapping with six highly conserved 75-bp units. The repetitive structure of the algP gene appears to participate in the processes underlying the metastable character of mucoidy in P. aeruginosa. Relatively large DNA rearrangements spanning the region with tandem direct repeats encoding the carboxy-terminal histone H1-like structure of AlgP were detected in several strains upon conversion from the mucoid to the nonmucoid phenotype. The frequency of the detectable algP rearrangements associated with the transition into the nonmucoid state varied from strain to strain and ranged from 0 to 50%. The nonmucoid derivatives with the clearly rearranged chromosomal copy of algP were complemented to mucoidy with plasmids containing algP from P. aeruginosa PAO. When a random collection of mucoid strains, isolated from different cystic fibrosis patients, was analyzed by using polymerase chain reaction, an additional level of strain-dependent sequence variation in algP was observed. Variations in the number of the 12-bp repeats were found; however, they did not appear to influence the mucoid status of the strains examined. Thus, the repeated region of algP appears to be a hot spot for DNA rearrangements and strain-dependent variability.

Amino Acid Sequence↗

PubMed1990

Gene-scrambling mutagenesis: generation and analysis of insertional mutations in the alginate regulatory region of Pseudomonas aeruginosa.

A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and lambda packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algR gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulatory of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source.

Alginates↗

PubMed1990

Role of the far-upstream sites of the algD promoter and the algR and rpoN genes in environmental modulation of mucoidy in Pseudomonas aeruginosa.

The role of several regulatory elements in environmental modulation of mucoidy in Pseudomonas aeruginosa was studied. Transcriptional activation of algD, necessary for the mucoid phenotype, was found to depend on FUS, the newly identified far-upstream sites of the algD promoter. The FUS were delimited to a region spanning nucleotides -432 to -332 relative to the algD mRNA start site. Insertional inactivation of algR in PAO568 abolished the algD promoter response to nitrogen availability and greatly diminished but did not completely eliminate reactivity to changes in salt concentration. Insertional inactivation of rpoN (ntrA) in PAO568 did not affect algR and algD transcription.

Base Sequence↗

PubMed1990

DNA sequence and expression analysis of algP and algQ, components of the multigene system transcriptionally regulating mucoidy in Pseudomonas aeruginosa: algP contains multiple direct repeats.

The complete nucleotide sequence of a 3.2-kilobase-pair chromosomal region containing the algP and algQ genes was determined. The algQ gene encodes an acidic 18-kilodalton polypeptide required for transcriptional activation of the algD gene. The algD gene product catalyzes a critical step in alginate biosynthesis, and its overproduction is necessary for the emergence of mucoid Pseudomonas aeruginosa during chronic infections in cystic fibrosis. A novel genetic element, algP, was identified immediately downstream of algQ. This gene appears to act synergistically with algQ. Unlike a biosynthetic gene, algD, and another regulatory gene, algR, which undergo transcriptional activation in mucoid cells, both algP and algQ are equally transcribed in mucoid and nonmucoid isogenic strains of P. aeruginosa. The promoter regions of algP and algQ were mapped by using S1 nuclease protection analysis. The algQ promoter was also analyzed and showed activity in an in vitro transcriptional runoff assay with major RNA polymerase species from P. aeruginosa and Escherichia coli. The putative algQ and algP promoter sequences, unlike algD and algR, resemble sigma 70-utilized promoters from E. coli and appeared constitutively transcribed at a low level in P. aeruginosa. The algP gene has an unusual DNA sequence, with multiple direct repeats organized in six highly conserved, tandemly arranged, 75-base-pair (bp) units. At a lower level, this sequence had 45 degenerate repeats of 12 bp overlapping with the 75-bp repeats and extending beyond the region of 75-bp repeats. The algP repeats appeared important for the function of the algQ-algP regulatory region in maintaining mucoidy.

Alginates↗

PubMed1989

The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes.

The Pseudomonas aeruginosa capsule, composed of polysaccharide alginate, is an important Pseudomonas virulence factor encountered primarily in cystic fibrosis. The regulatory algR gene positively controls transcription of a key alginate biosynthetic gene, algD. The algR gene was subcloned and sequenced by creating a set of nested deletions in M13 bacteriophage. DNA sequence analysis of algR revealed the homology of its gene product with a recently recognized class of environmentally responsive bacterial regulatory genes, including ompR, phoB, sfrA, ntrC, spoOA, dctD, and virG; these transcriptional activators control cellular reactions to osmotic pressure, phosphate limitations, or specific chemical compounds present in the medium or released from wounded host tissue. These findings indicate that novel conditions in lungs affected by cystic fibrosis may be participating in the control of mucoidy.

Alginates↗

PubMed1989

Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ.

A new alginate regulatory gene, algQ, was identified in a chromosomal region which, when tandemly amplified, induces mucoidy in Pseudomonas aeruginosa. The algQ gene was found closely linked to the previously identified algR gene. Both algQ and algR were required for transcription of the key alginate biosynthetic gene, algD. In addition, expression of the algR gene was studied. The algR promoter was mapped by S1 nuclease and reverse transcription and found to be activated in mucoid cells. However, even in nonmucoid cells, transcription of algR was detectable at an approximately 50-fold-lower level, as opposed to the algD promoter, which was silent in the nonmucoid background. Transcription of both promoters was studied by using algR- and algD-specific oligonucleotides and total cellular RNA from fresh cystic fibrosis isolates of mucoid P. aeruginosa and their nonmucoid revertants. Identical patterns of activity were found in all strains: in mucoid cells, both algR and algD were activated. This finding indicated that common mechanisms were involved in the regulation of alginate gene expression. However, when the algR gene was cloned behind the tac promoter on a broad-host-range-controlled expression vector, induction of transcription with isopropropyl-beta-D-thiogalactopyranoside (IPTG) caused the appearance of a nonmucoid phenotype in previously mucoid cells. This effect was transient, since removal of the inducer (IPTG) made cells mucoid again. Since the algR gene product is homologous to transcriptional regulators from a class of environmentally responsive systems (known to have a second, sensory component), the algQ gene could be a candidate for the sensory component of the alginate system.

Alginates↗

PubMed1988

Broad-host-range plasmid and M13 bacteriophage-derived vectors for promoter analysis in Escherichia coli and Pseudomonas aeruginosa.

A set of bacteriophage and plasmid vectors containing xylE as a reporter gene was constructed for the analysis of promoters functional in Escherichia coli and in other Gram-negative bacteria. Two M13 bacteriophage derivatives, M13mVDX18 and M13mMK010, were designed for rapid cloning, screening and sequencing of DNA fragments promoting transcription in E. coli. To demonstrate their utility, total cellular DNA from a variety of bacterial species including Pseudomonas aeruginosa strain PAO was shotgun cloned in M13 vectors and clones displaying promoter activity in E. coli were isolated. These randomly cloned promoters from P. aeruginosa, Borrelia burgdorferi, Streptococcus pneumoniae and other bacterial species were sequenced without a need for further subcloning manipulation. The promoter activity of P. aeruginosa clones was verified by subcloning inserts on a broad-host-range promoter probe vector pVDX18 and assaying the xylE transcription from these promoters in P. aeruginosa. The pVDX18 vector was also used for initial characterization of the algD promoter controlling mucoidy in P. aeruginosa. The activities of the wild-type and deletion clones of the algD promoter were compared. Results indicated that the region containing direct and inverted repeats at -55 to -110 bp upstream of the mRNA 5' end was important for the activation of the algD transcription in mucoid P. aeruginosa infecting cystic fibrosis patients.

Bacteriophages↗