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A Darzins

Publications and source records attributed to A Darzins.

At least 19 recordsLinked to original sources
PubMed2001

DNA shuffling method for generating highly recombined genes and evolved enzymes.

We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.

Alleles↗
PubMed1997

Molecular genetic analysis of type-4 pilus biogenesis and twitching motility using Pseudomonas aeruginosa as a model system--a review.

Genetic analysis of Pseudomonas aeruginosa pilus biogenesis and twitching motility has revealed the requirement for several pil loci which have been localized to different regions of the chromosome. One pil locus, designated pilE, resides at approx. 71 min on the PAO genetic map, a region of the chromosome previously shown to harbor a number of genes required for pilus assembly (i.e., pilA, -B, -C, -D, -R and -S). The PilE protein shows significant sequence identity to the N-terminal domain of PilA as well as to the pilin precursors from a variety of type-4 pilus producers. Included within this homologous region is a short, positively charged leader sequence followed by a prepilin peptidase cleavage site and a largely hydrophobic region. Additionally, an unlinked set of pil genes, designated pilG, -H, -I, -J and -K, has been localized to the SpeI fragment H which corresponds to approx. 20 min on the PAO genetic map. This gene cluster encodes proteins that demonstrate remarkable similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus and are thought to be part of a signal transduction system that controls P. aeruginosa pilus biosynthesis and twitching motility.

Bacterial Outer Membrane Proteins↗
PubMed1997

Snr, new genetic loci common to the nitrate reduction systems of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is able to both assimilate and dissimilate nitrate. On the basis of the characteristics of mutants unable to dissimilate or assimilate nitrate to nitrite, it was revealed that two different sets of genes (represented by Class I and Class II mutants) were shared between the nitrate-to-nitrite reduction steps of both pathways. The genes represented by Class I and II mutants have been separated into distinct genetic loci using two cosmids, pAD1695/96. The two different genetic loci have been designated snr (shared nitrate reduction) and mol (MoCo processing genes) based on the phenotypic characteristics of the mutants complemented. Restriction analyses of pAD1695/96 followed by subcloning confirmed the complementation results. The snr loci, which represent a unique and hitherto uninvestigated set of genes for nitrate reduction, were mapped on the P. aeruginosa chromosome by linkage analysis with sex factor FP2.

Chromosome Mapping↗
PubMed1995

The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated.

A new locus, designated pilK, located immediately adjacent to the previously described Pseudomonas aeruginosa pilG-J gene cluster, has been identified. Sequence analysis of a 1.3 kb region revealed the presence of a single open reading frame of 291 amino acid residues (M(r) 33,338) that contained significant homology to the chemotactic methyltransferase proteins of Escherichia coli, Bacillus subtilis and the gliding bacterium Myxococcus xanthus. The 60 bp pilJ-pilK intergenic region was devoid of promoter consensus sequences, suggesting that pilJ and pilK are contained within the same transcriptional unit. The intergenic region did contain, however, a large, highly GC-rich, inverted repeat that prevented PilK production in expression studies. To investigate the regulatory role of these sequences, pilK-lacZ gene fusions, as well as derivatives containing sequence alterations in the potential stem-loop region, were constructed and analysed in E. coli and P. aeruginosa. Modification of the inverted repeat region in pilK-lacZ protein fusion constructs resulted in as much as a 24-fold increase in beta-galactosidase activity, whereas similar modifications in pilK-lacZ transcriptional fusions had only a marginal effect on beta-galactosidase levels. These results indicated that PilK production may be largely regulated at the level of translation. In stark contrast to pilG-J mutants, which are dramatically impaired in pilus production and/or function, a PAO1 pilK deletion mutant was indistinguishable from the wild type. In addition, complementation studies suggested that the PilK and E. coli CheR proteins are not functionally interchangeable.

Amino Acid Sequence↗
PubMed1994

Characterization of a Pseudomonas aeruginosa gene cluster involved in pilus biosynthesis and twitching motility: sequence similarity to the chemotaxis proteins of enterics and the gliding bacterium Myxococcus xanthus.

The type 4 pili of Pseudomonas aeruginosa are important cell-associated virulence factors that play a crucial role in mediating (i) bacterial adherence to, and colonization of, mucosal surfaces, (ii) a novel mode of flagella-independent surface translocation known as 'twitching motility', and (iii) the initial stages of the infection process for a number of bacteriophages. A new set of loci involved in pilus biogenesis and twitching motility was identified based on the ability of DNA sequences downstream of the pilG gene to complement the non-piliated (pil) strain, PAO6609. Sequence analysis of a 3.2 kb region directly downstream of pilG revealed the presence of three genes, which have been designated pilH, pilI, and pilJ. The predicted translation product of the pilH gene (13,272 Da), like PilG, exhibits significant amino acid identity with the enteric single-domain response regulator CheY. The putative PilI protein (19,933 Da) is 28% identical to the FrzA protein, a CheW homologue of the gliding bacterium Myxococcus xanthus, and the PilJ protein (72,523 Da) is 26% identical to the enteric methyl-accepting chemotaxis protein (MCP) Tsr. Mutants containing insertions in pilI and pilJ were severely impaired in their ability to produce pili and did not translocate across solid surfaces. The pilH mutant remained capable of pilus production and twitching motility, but displayed an altered motility pattern characterized by the presence of many doughnut-shaped swirls. Each of these pil mutants, however, produced zones that were at least as large as the parent in flagellar-mediated swarm assays. The sequence similarities between the putative pilG, H, I and J gene products and several established chemotaxis proteins, therefore, lend strong support to the hypothesis that these proteins are part of a signal-transduction network that controls P. aeruginosa pilus biosynthesis and twitching motility.

Amino Acid Sequence↗
PubMed1994

The pilE gene product of Pseudomonas aeruginosa, required for pilus biogenesis, shares amino acid sequence identity with the N-termini of type 4 prepilin proteins.

A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ-6, was broadly resistant to pili-specific phages and unable to translocate across solid surfaces by the pilus-dependent mechanism of twitching motility (Twt-). Immunoblot analysis demonstrated that MJ-6 was devoid of pili (Pil-) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing the pilE mutation on the P. aeruginosa PAO chromosome demonstrated a strong co-linkage between MJ-6 phage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a pro(B+)-containing plasmid from a PAO1 cosmid library. Upon introduction of the PAO1 proB+ cosmid clone into MJ-6, sensitivity to pili-specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kb EcoRV-ClaI fragment containing the pilE region revealed a single complete open reading frame with characteristic P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15,278, showed significant sequence identity to the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. The region of highest homology was localized to the N-terminal 40 amino acid residues. The putative PilE N-terminus contained a seven-residue basic leader sequence followed by a consensus cleavage site for prepilin peptidase and a largely hydrophobic region which contained tyrosine residues (Tyr-24 and Tyr-27) previously implicated in maintaining pilin subunit-subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomal pilE insertion mutants were pilus- and twitching-motility deficient.

Amino Acid Sequence↗
PubMed1993

Pseudomonas aeruginosa lasA gene: determination of the transcription start point and analysis of the promoter/regulatory region.

The elastolytic activity of the opportunistic pathogen Pseudomonas aeruginosa is due to the combined activities of at least three secreted proteins: elastase, LasA and alkaline protease. Transcription of both the lasA gene and the elastase structural gene, lasB, requires the transcriptional activator LasR. In order to localize the promoter elements involved in lasA expression, the transcription start point (tsp) for lasA was localized by S1 protection and primer extension analysis. The DNA sequence of the region upstream from the tsp was determined, and a putative sigma promoter was identified. Sequence comparison with the lasB promoter region revealed two areas of considerable homology which could act as potential binding sites for LasR or other, as yet unidentified, regulatory proteins.

Bacterial Proteins↗
PubMed1993

The pilG gene product, required for Pseudomonas aeruginosa pilus production and twitching motility, is homologous to the enteric, single-domain response regulator CheY.

The Pseudomonas aeruginosa pilG gene, encoding a protein which is involved in pilus production, was cloned by phenotypic complementation of a unique, pilus-defective mutant of strain PAO1. This mutant, designated FA2, although resistant to the pilus-specific phage D3112 was sensitive to the pilus-specific phages B3 and F116L. In spite of the unusual phage sensitivity pattern, FA2 lacked the ability to produce functional polar pili (pil) and was incapable of twitching motility (twt). Genetic analysis revealed that the FA2 pil mutation, designated pilG1, mapped near the met-28 marker located at 20 min and was distinct from the previously described pilT mutation. This map location was confirmed by localization of a 6.2-kb EcoRI fragment that complemented FA2 on the SpeI and DpnI physical map of the P. aeruginosa PAO1 chromosome. A 700-bp region encompassing the pilG gene was sequenced, and a 405-bp open reading frame, with characteristic P. aeruginosa codon bias, was identified. The molecular weight of the protein predicted from the amino acid sequence of PilG, which was determined to be 14,717, corresponded very closely to that of a polypeptide with the apparent molecular weight of 15,000 detected after expression of pilG from the T7 promoter in Escherichia coli. Moreover, the predicted amino acid sequence of PilG showed significant homology to that of the enteric CheY protein, a single-domain response regulator. A chromosomal pilG insertion mutant, constructed by allele replacement of the wild-type gene, was not capable of pilus production or twitching motility but displayed normal flagellum-mediated motility. These results, therefore, suggest that PilG may be an important part of the signal transduction system involved in the elaboration of P. aeruginosa pili.

Amino Acid Sequence↗
PubMed1992

A two-component T7 system for the overexpression of genes in Pseudomonas aeruginosa.

A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aeruginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUV5/lacIq-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This element, designated miniD-180, was stably integrated into the P. aeruginosa PAO1 chromosome. The second component of this system includes several improved broad-host-range expression vectors containing the T7 gene 10 promoter and multiple cloning site (MCS). These vectors (pEB8, pEB11, and pEB12) contain transcriptional terminators (T1(4)) upstream from the T7 promoter, and T7 terminators downstream from the MCS. Because the T7 promoter is somewhat leaky in these vectors, pEB14 was constructed to decrease transcription of target genes by basal levels of T7 RNA polymerase. This vector contains a core sequence of the lac operator located 19 bp downstream from the transcriptional start point of the T7 promoter, thereby providing a dually regulated system. The utility of this system was demonstrated by placing a promoterless chloramphenicol acetyltransferase (CAT) cassette under control of the T7 promoter and monitoring the isopropyl-beta-D-thiogalactopyranoside-dependent accumulation of CAT in cell-free extracts of P. aeruginosa. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction, at which time this polypeptide represented up to 20% of the total soluble protein.

Chloramphenicol O-Acetyltransferase↗
PubMed1992

Further studies on Pseudomonas aeruginosa LasA: analysis of specificity.

Full elastolytic activity in Pseudomonas aeruginosa is a result of the combined activities of elastase, alkaline proteinase, and the lasA gene product, LasA. The results of this study demonstrate that an active fragment of the LasA protein which is isolated from the culture supernatant fraction is capable of degrading elastin in the absence of elastase, thus showing that LasA is a second elastase produced by this organism. In addition, it is shown that LasA-mediated enhancement of elastolysis results from the separate activities of LasA and elastase upon elastin. The LasA protein does not affect the secretion or activation of a proelastase as previously proposed in other studies. Furthermore, LasA has specific proteolytic capability, as demonstrated by its ability to cleave beta-casein. Preliminary analysis of beta-casein cleavage in the presence of various protease inhibitors suggests that LasA may be classified as a modified serine protease.

Amino Acid Sequence↗
PubMed1991

Imipenem resistance in pseudomonas aeruginosa PAO: mapping of the OprD2 gene.

Carbapenem antibiotics have been shown to penetrate the outer membrane of Pseudomonas aeruginosa through a unique porin protein, OprD2. We mapped the OprD2 gene by conjugation using plasmid FP2 and by transduction using phage F116L. This gene maps between 71 and 75 min on the PAO1 chromosome.

Bacterial Outer Membrane Proteins↗
PubMed1990

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 require pili and surface growth for adsorption.

Pseudomonas aeruginosa transposable bacteriophages D3112 and B3 were found to require pili for infection. Seventy mutants of P. aeruginosa PAO selected by resistance to D3112 or B3 were also resistant to the phage not used in the selection and suggested that the receptors of these two phages are identical. Of five resistant mutants examined, all were defective in the production of pili and did not adsorb either phage. P. aeruginosa PAK strains altered in pilus expression, such as hyperpiliated or nonpiliated mutants, adsorbed the phage but were not productively infected, implying that an additional host function was required for infection. The cell-associated lipopolysaccharide was not required for D3112 or B3 infection, since mutants deficient in O side-chain and core biosynthesis were still capable of adsorption and productive infection. This is in contrast to Escherichia coli mutator phages Mu and D108, which are dependent on lipopolysaccharide for adsorption. The P. aeruginosa phages adsorbed only to cells grown on solid media or in liquid media supplemented with agents that increase the macroviscosity, such as polyvinylpyrrolidone. Adsorption time course studies of D3112 and B3 using cells grown in solid media revealed similar but not identical adsorption patterns. These studies suggested that expression of the D3112 and B3 cell receptor is induced by growth on solid media.

Adsorption↗
PubMed1989

Mini-D3112 bacteriophage transposable elements for genetic analysis of Pseudomonas aeruginosa.

Small bacteriophage D3112 transposable elements deleted for most of the phage-lytic functions while retaining the sites required for transposition and packaging were constructed to facilitate genetic studies in Pseudomonas aeruginosa. These mini-D derivatives were constructed with the terminal 1.85 kilobases (kb) of the phage left end and 1.4 kb of the phage right end and either the Tn5 kanamycin resistance or the pSC101 (pBR322) tetracycline resistance determinant. Thermally induced lysates of strains lysogenic for both a mini-D element and D3112 cts (temperature-sensitive repressor) transduced P. aeruginosa PAO recipients to drug resistance at frequencies of between 10(-4) and 10(-5)/PFU of the helper phage. As for the parent plaque-forming D3112 phage, the mini-D171 element could insert itself into many different sites in the chromosome but the frequency of insertion into particular genes varied widely. Among 1,000 insertions, none resulted in auxotrophy but 10 resulted in pigment production. Insertions were also selected in a cloning plasmid with a transduction scheme. At least eight different insertion sites were found to have been used among 10 individual insertions. Transductants harboring these mini-D elements were immune to infection by D3112, since they contained the D3112 repressor gene in the left 1.85-kb terminal fragment. Chromosomal genes were transduced in a generalized fashion 100 to 1,000 times more frequently by the mini-D-D3112 cts lysates than by the D3112 cts phage alone. Mini-D171-D3112 cts lysates also yielded some transductants that retained the drug resistance marker of the mini-D element and which were unstable for the chromosomal transduced marker. This is consistent with the miniduction properties of Mu whereby transduced genes are flanked by two mini-D elements in the same orientation.

Bacteriophages↗