Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated. 778 42

Reporter gene technology was used to observe the regulation of the alginate biosynthesis gene, algC in a mucoid strain of Pseudomonas aeruginosa in developing and mature biofilms in continuous culture on Teflon and glass substrata. The plasmid pNZ63, carrying an algC-lacZ transcriptional fusion, was shown to not be diluted in continuous culture over a period of 25 days in the absence of selection pressure. Biofilm cells under bulk phase steady-state conditions demonstrated fluctuations in algC expression over a 16-day period, but no trend of increased or decreased expression over the time interval was indicated. In vivo detection of algC up-expression in developing biofilms was performed with a fluorogenic substrate for the plasmid-borne lacZ gene product (beta-galactosidase) by using microscopy coupled with image analysis. By this technique, cells were tracked over time and analyzed for algC activity. During the initial stages of biofilm development, cells already attached to a glass surface for at least 15 min exhibited up-expression of algC, detectable as the development of whole-cell fluorescence. However, initial cell attachment to the substratum appeared to be independent of algC promoter activity. Furthermore, cells not exhibiting algC up-expression were shown to be less capable of remaining at a glass surface under flowing conditions than were cells in which algC up-expression was detected.
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PMID:Regulation of the alginate biosynthesis gene algC in Pseudomonas aeruginosa during biofilm development in continuous culture. 779 20

The multimer resolution system (mrs) of the broad-host-range plasmid RP4 has been exploited to develop a general method that permits the precise excision of chromosomal segments in a variety of gram-negative bacteria. The procedure is based on the site-specific recombination between two directly repeated 140-bp resolution (res) sequences of RP4 effected by the plasmid-borne resolvase encoded by the parA gene. The efficiency and accuracy of the mrs system to delete portions of chromosomal DNA flanked by res sites was monitored with hybrid mini-Tn5 transposons in which various colored (beta-galactosidase and catechol 2,3 dioxygenase) or luminescent (Vibrio harveyi luciferase) phenotypic markers associated to res sequences were inserted in the chromosome of the target bacteria and exposed in vivo to the product of the parA gene. The high frequencies of marker excision obtained with different configurations of the parA expression system suggested that just a few molecules of the resolvase are required to achieve the site-specific recombination event. Transient expression of parA from a plasmid unable to replicate in the target bacterium was instrumental to effect differential deletions within complex hybrid transposons inserted in the chromosome of Pseudomonas putida. This strategy permits the stable inheritance of heterologous DNA segments virtually devoid of the sequences used initially to select their insertion.
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PMID:Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4. 779 49

Pseudomonas aeruginosa PAO was mutagenized with Tn1737KH, a type I transcription probe transposon containing a promoterless lacZ (beta-galactosidase) gene, and 24 insertion mutants that did not grow under iron-deficient conditions were isolated. None of the culture supernatants from any mutants contained pyoverdin, a low-molecular-weight siderophore able to sequester ferric iron at very high affinity, and the growth defects of the mutants were all phenotypically recovered by the addition of the culture supernatant from the wild-type strain. These phenotypes led to the inference that all the mutants had defects in the genes (pvd genes) for production of pyoverdin. In some pvd::Tn1737KH mutants, high levels of beta-galactosidase activities were observed, and such activities were drastically reduced by the addition of ferric ion in the culture media, indicating that the expression of at least some pvd genes is regulated at the transcriptional level. Molecular cloning and physical analysis of the chromosomal fragments with Tn1737KH insertions allowed us to allocate all the mutations within a 103-kb region, referred to as the pvd region, that was found to locate at 47 min on the genetic map of PAO. Further physical mapping and Southern analysis showed that there is a 10-kb overlap between the pvd region and the 125-kb catA region described by Zhang and Holloway (C. Zhang and B. W. Holloway, J. Gen. Microbiol. 138:1097-1107, 1992). We could hence illustrate the physical map of the P. aeruginosa chromosome with a size of 218 kb.
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PMID:Genetic and physical mapping of genes involved in pyoverdin production in Pseudomonas aeruginosa PAO. 781 32

In Pseudomonas aeruginosa, the transcriptional activator LasR and the Pseudomonas autoinducer PAI, are necessary for efficient transcriptional activation of the lasB gene, encoding elastase (L. Passador, J. M. Cook, M.J. Gambello, L. Rust, and B. H. Iglewski, Science 260:1127-1130, 1993). The transcriptional start points of lasI in Escherichia coli and P. aeruginosa were determined by S1 nuclease mapping. In the presence of both LasR and PAI, the start site, T1, is located at position -25 relative to the ATG translational start codon. A minor transcriptional start, T2, is found at position -13 when lasI is transcribed in the absence of either LasR or PAI in P. aeruginosa and E. coli, respectively. To begin to closely examine the regulation of lasI, whose product is involved in the synthesis of PAI, a lasI-lacZ fusion on a lambda phage was constructed to form monolysogens of E. coli MG4. Lysogens supplied only with either lasI or lasR via multicopy plasmids demonstrated no significant increase in beta-galactosidase expression compared with control levels. Lysogens in which both lasR and lasI were supplied in multicopy exhibited a 62-fold increase in expression, and a lysogen in which lasR was supplied in trans and which was grown in the presence of exogenous PAI exhibited a 60-fold increase. Thus, LasR and PAI are necessary for the full expression of lasI in E. coli. The interchangeability of the P. aeruginosa and Vibrio fischeri homologs LasR and LuxR and their respective autoinducers, PAI and VAI, as activators of lasI-lacZ was examined. Only the combination of LasR and PAI significantly increased the expression of lasI. The comparison of lasI-lacZ and lasB-lacZ expression lysogens grown in the presence of lasR and PAI revealed that half-maximal expression of lasI required 0.1 nM PAI, in contrast to the 1.0 nM PAI necessary for lasB half-maximal expression. These results suggest an autoinduction regulatory hierarchy in which LasR and low PAI concentrations primarily activate lasI expression in a regulatory loop. With the accumulation of PAI, secondary activation of virulence product genes such as lasB occurs.
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PMID:Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. 783 99

Escherichia coli LE392 (pAL28) was previously isolated as a positive clone harboring the alginate lyase gene (aly) from an alginate-degrading strain, Pseudomonas sp. OS-ALG-9. The plasmid pAL205, one of the constructs obtained after successive subcloning of pAL28, gave the highest expression of aly in E. coli cells. A 8-fold increase in the alginate lyase (Aly) activity in E. coli JM109 (pAL205) was induced with isopropyl-beta-D-thiogalactoside, which was 210 times higher than that in E. coli LE392 (pAL28). The highly significant increase in the expression of the Aly enzyme with pAL205 was investigated through the nucleotide sequence around the 5' region of aly as well as the N-terminal sequence of the purified enzyme. It was found that the Aly expressed in E. coli (pAL205) was a fused protein containing 7 residues from the N-terminus of beta-galactosidase alpha-peptide and the mature protein found in the Pseudomonas sp. except for three residues in the N-terminal.
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PMID:High gene expression in Escherichia coli of recombinant alginate lyase as a fused protein with beta-galactosidase alpha-peptide. 789 71

The plasmid pAL205 encodes an alginate lyase gene of Pseudomonas sp. OS-ALG-9, fused in frame to the beta-galactosidase alpha-peptide gene. The alginate lyase (Aly) expressed in Escherichia coli (pAL205) was significantly secreted into the medium by the addition of glycine. The extracellular enzyme isolated from the culture of E. coli JM109 (pAL205) was purified over 15,000-fold by successive chromatography and subjected to amino acid sequence analysis. The sequence determined was identical to that of the intracellular protein. Since the activity and molecular size of the extracellular Aly is identical to the intracellular protein and to the Aly isolated from Pseudomonas, the glycine does not affect or modify the Aly during its leakage into the medium.
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PMID:Purification and characterization of the recombinant alginate lyase from Pseudomonas sp. leaked by Escherichia coli upon addition of glycine. 789 72

The cytochrome P450cam hydroxylase operon (camDCAB) of Pseudomonas putida is negatively regulated by a repressor, CamR, which also represses its own gene. The expression in P. putida of both camR and camDCAB is derepressed in the presence of D-camphor. We examined the expression in Escherichia coli of camR and camDCAB by monitoring the enzyme activity of the camD gene product. In the presence or absence of D-camphor in the cell culture, the expression in E. coli of camD was significant and constitutive, suggesting no expression of camR. This lack of expression was confirmed by monitoring the beta-galactosidase activity of camR-lacZ translational fusions. However, S1 nuclease mapping revealed that synthesis of camR mRNA in E. coli was significant and constitutive, as observed in the case of camDCAB mRNA. Thus, it is likely that the expression of camR in E. coli is limited at the translational level.
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PMID:Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli. 798 98

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.
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PMID:The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. 800 77

The algC gene (encoding phosphomannomutase) of Pseudomonas aeruginosa, similarly to the algD gene, is environmentally regulated through transcriptional activation of its promoter. This gene, like algD, has a long (244 bp) 5' untranslated leader region (5' UTR). Using transcriptional and translational algC::lacZ fusions, we show that even though the transcript levels are similar, the beta-galactosidase-specific activities of the translational fusions are much higher than those of the transcriptional fusions during the entire growth phase. Both the 5' UTR and the ribosomal-binding site are shown to be important for efficient translation of the algC mRNA.
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PMID:Post-transcriptional regulation of the Pseudomonas aeruginosa algC gene. 806 91


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