Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

We cloned and sequenced a 2.8-kb SalI fragment of Azospirillum lipoferum FS as a homologue of the Klebsiella oxytoca nifA gene. The amino acid sequence deduced from an open reading frame of 1872 bases showed 91% identity to that of the A. brasilense NifA, and the putative central sigma54 interaction domain was conserved as well as the C-terminal DNA-binding domain. The NifA function on the nifH promoter was examined in Escherichia coli using a combination of a nifA driver plasmid and a nifH-lacZ reporter plasmid, in which the transcriptional activation of the nifH promoter by the NifA was evaluated with the beta-galactosidase activity. The A. lipoferum NifA activated the nifH promoter solely under microaerobic conditions, while the K. oxytoca NifA activated it irrespective of the oxygen condition. These observations suggest that oxygen sensitivity is an intrinsic property of the A. lipoferum NifA.
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PMID:Oxygen sensitivity of NifA protein of Azospirillum lipoferum FS as suggested by gene cloning and expression in Escherichia coli. 917 50

From Azospirillum brasilense Yu62, we cloned and sequenced the upstream region of draTG. No identified genes were found in this region except for portion of nifH, which is transcribed divergently from draTG. However, some potential regulatory elements were found, which include downstream promoter elements (DPE), upstream activator sequences (UAS), and A + T-rich regions. This suggests that the region between draT and nifH might be a regulatory region rather than an encoding region, and the promoter of dra operon would probably be a RpoN-dependent promoter. A draT::cam transcriptional fusion plasmid pAT1 was constructed in pAF300. The expression of draT was studied in Escherichia coli and A. brasilense by Cmr assay. The result showed that draT could be transcribed in A. brasilense but not in E. coli while grown aerobically on an LD plate. This suggests that the transcription of draT needs some factors which are absent in E. coli, and the draT upstream region has the promoter function. Using the promoter-probe vector pCB182, a draT::lacZ transcriptional fusion plasmid pCT1 was constructed. beta-galactosidase activity was determined in vivo in E. coli, in the presence or absence of NifA of Klebsiella pneumoniae, respectively. The results demonstrated that NifA was not involved in the transcriptional regulation of draTG.
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PMID:Sequencing and analysis of function of the promoter region of draTG genes from Azospirillum brasilense Yu62. 963 Dec 55

The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from -53 to +27 and the region around -116 containing an ihf binding site. Both beta-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.
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PMID:Identification and characterization of the fis operon in enteric bacteria. 981 52

Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon. Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of nasR gene expression. We constructed strains bearing single-copy Phi(nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus. The expression of DeltarhaBS::[Phi(nasR-lacZ)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma(N) (sigma(54)) proteins. Sequence inspection of the nasR control region reveals an apparent sigma(N)-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma(N)-dependent nasR promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta-galactosidase expression indistinguishable from Phi(nasR-lacZ) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription. Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107-117, 1988).
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PMID:General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al. 1057 31

A hybrid promoter consisting of the in tandem fusion of the Tn5 nptII and the Klebsiella pneumoniae nifH promoters was constructed to study the functionality of the nif genes transcriptional activator NifA from Bradyrhizobium japonicum in two different host bacteria. beta-galactosidase experiments in E. coli revealed that the hybrid nptII-nifH promoter can behave as a constitutive or a NifA-inducible promoter depending on the aeration conditions. Expression of the B. japonicum NifA from the hybrid nptII-nifH promoter (plasmid pBPF204) induced "in trans" lacZ transcription from the Azotobacter chroococcum nifH promoter in E. coli and A. diazotrophicus cells grown at low pO2. Similarly, the plasmid pBPF204 increased nitrogenase activity in A. diazotrophicus cells grown under microaerobic conditions. Based on these results, we suggest that the B. japonicum NifA could function as an efficient O2-sensitive transcriptional activator of nif genes in genetically distant diazotrophic bacteria.
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PMID:Functional Bradyrhizobium japonicum NifA expression under a hybrid nptII-nifH promoter in E. coli and Acetobacter diazotrophicus SRT4. 1093 42

The gene encoding beta-galactosidase was isolated by functional complementation of Escherichia coli from Bifidobacterium longum MB219, which exhibited the highest activity among ten Bifidobacterium strains tested of the species B. longum, B. breve, B. adolescentis, B. indicum, B. animalis and B. cuniculi. The nucleotide sequence of the 5.0-kb fragment conferring the positive beta-galactosidase phenotype to E. coli revealed the presence of a lacZ-type gene encoding a 1023-amino-acid protein that was preceded by a ribosome binding site. A sequence showing 72% identity with the proline tRNA of Bacillus subtilis and a gene probably encoding the DNA-3-methyladenine glycosydase I were located downstream from the lacZ gene, after a gap of 30-50 unsequenced base pairs. By primer-extension analysis, the transcription start site of the lacZ gene was mapped 65 nt upstream from the start codon, and it enabled identification of the -10 region of the putative promoter. The nucleotide sequence of lacZ and its deduced amino acid sequence were compared with those of beta-galactosidase genes and enzymes from other microorganisms. High similarity was demonstrated between the B. longum beta-galactosidase and its counterparts in Lactobacillus delbruckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus, E. coli, Clostridium acetobutylicum, Leuconostoc lactis, Klebsiella pneumoniae and Kluyveromyces marxianus var. lactis, all belonging to the LacZ family. The B. longum MB219 lacZ gene was cloned in Bifidobacterium and its expression was observed in strains with otherwise low levels of endogenous activity. The expression increased by factors of 1.5-50 and enabled those strains that do not grow on lactose to use this sugar as sole carbon source.
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PMID:Nucleotide sequence, expression and transcriptional analysis of the Bifidobacterium longum MB 219 lacZ gene. 1098 45

The quantitative determination of total and fecal coliforms, as indicators of fecal pollution, is essential for water quality control. We developed a sensitive, inexpensive amperometric enzyme biosensor based on the electrochemical detection of beta-galactosidase activity, using p-amino-phenyl-beta-D-galactopyranoside as substrate, for determining the density of coliforms, represented by Escherichia coli and Klebsiella pneumoniae. The specific detection of E. coli was achieved using an antibody-coated electrode that specifically binds the target bacteria. Amperometric detection enabled the determination of 1000 colony-forming units/mL within 60-75 min. Preincubation for 5-6 h further increased the sensitivity more than 100-fold. The present experimental setup allowed the simultaneous analysis of up to eight samples, using disposable screen-printed electrodes.
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PMID:Amperometric quantification of total coliforms and specific detection of Escherichia coli. 1186 71

The intracellular concentration of the Escherichia coli factor for inversion stimulation (Fis), a global regulator of transcription and a facilitator of certain site-specific DNA recombination events, varies substantially in response to changes in the nutritional environment and growth phase. Under conditions of nutritional upshift, fis is transiently expressed at very high levels, whereas under induced starvation conditions, fis is repressed by stringent control. We show that both of these regulatory processes operate on the chromosomal fis genes of the enterobacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris, strongly suggesting that the physiological role of Fis is closely tied to its transcriptional regulation in response to the nutritional environment. These transcriptional regulatory processes were previously shown to involve a single promoter (fis P) preceding the fis operon in E. coli. Recent work challenged this notion by presenting evidence from primer extension assays which appeared to indicate that there are multiple promoters upstream of fis P that contribute significantly to the expression and regulation of fis in E. coli. Thus, a rigorous analysis of the fis promoter region was conducted to assess the contribution of such additional promoters. However, our data from primer extension analysis, S1 nuclease mapping, beta-galactosidase assays, and in vitro transcription analysis all indicate that fis P is the sole E. coli fis promoter in vivo and in vitro. We further show how certain conditions used in the primer extension reactions can generate artifacts resulting from secondary annealing events that are the likely source of incorrect assignment of additional fis promoters.
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PMID:Growth phase-dependent regulation and stringent control of fis are conserved processes in enteric bacteria and involve a single promoter (fis P) in Escherichia coli. 1467 32

From a mixture of N-acetylglucosaminyl-beta-cyclodextrin (GlcNAc-betaCD) and lactose, beta-D-galactosyl-GlcNAc-betaCD (Gal-GlcNAc-betaCD) was synthesized by the transfer action of beta-galactosidase. GlcNAc-maltotriose (Glc3) and Gal-GlcNAc-Glc3 were produced with hydrolysis of GlcNAc-betaCD by cyclodextrin glycosyltransferase, and Gal-GlcNAc-betaCD by bacterial saccharifying alpha-amylase respectively. Finally, GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were synthesized in 5.2% and 3.5% yield when Klebsiella pneumoniae pullulanase was incubated with the mixture of GlcNAc-Glc(3) and betaCD, or Gal-GlcNAc-Glc3 and betaCD respectively. The structures of GlcNAc-Glc3-betaCD and Gal-GlcNAc-Glc3-betaCD were analyzed by FAB-MS and NMR spectroscopy and identified as 6-O-alpha-(6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD, and 6-O-alpha-(4-O-beta-D-galactopyranosyl-6(3)-O-beta-D-N-acetylglucosaminyl-maltotriosyl)-betaCD respectively.
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PMID:Synthesis of novel heterobranched beta-cyclodextrins having beta-D-N-acetylglucosaminyl-maltotriose on the side chain. 1584 11

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of "inverse" microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor sigma(28) was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed beta-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.
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PMID:Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions. 1659 95


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