Cloning and sequence analysis of the heat-stable acrylamidase from a newly isolated thermophilic bacterium, Geobacillus thermoglucosidasius AUT-01.

A thermophilic bacterium capable of degrading acrylamide, AUT-01, was isolated from soil collected from a hot spring area in Montana, USA. The thermophilic strain grew with 0.2 % glucose as the sole carbon source and 1.

Characterization of Acrylamidase isolated from a newly isolated acrylamide-utilizing bacterium, Ralstonia eutropha AUM-01.

A mesophilic bacterium capable of utilizing acrylamide was isolated, AUM-01, from soil collected from leaf litter at Picnic Point on the UW-Madison campus. In minimal medium with acrylamide as the sole carbon and nitrogen source, a batch culture of AUM-01 completely converted 28.0 mM acrylamide to acrylic acid in 8 h and reached a cell density of 0.

Evidence that Bacillus catabolite control protein CcpA interacts with RNA polymerase to inhibit transcription.

Summary Bacilluscatabolite control protein (CcpA) mediates carbon catabolite repression (CCR) by controlling expression of catabolite responsive (CR) genes or operons through interaction with catabolite responsive elements (cres) located within or outside of CR promoters. Here, we investigated how CcpA inhibits the transcription of CR promoters in vitro. CcpA has different affinities for different cres, but this does not correlate with its ability to inhibit transcription.

Crystallization and preliminary analysis of xenobiotic reductase B from Pseudomonas fluorescens I-C.

Single crystals have been obtained of xenobiotic reductase B (XenB), a flavoenzyme isolated and cloned from Pseudomonas fluorescens I-C. The enzyme catalyzes the NADPH-dependent elimination of nitrite from nitroglycerin with an approximately fivefold kinetic preference for the middle nitro group, primarily yielding 1,3-dinitroglycerol. X-ray diffraction data sets have been collected from native crystals to 2.

Crystallization and preliminary analysis of xenobiotic reductase A and ligand complexes from Pseudomonas putida II-B.

Diffraction-quality crystals have been obtained of the xenobiotic reductase A (XenA) from Pseudomonas II-B, which was originally cultured from the contaminated soil of a World War II era munitions-manufacturing plant. Several complete X-ray diffraction data sets have been collected and analyzed. The native XenA data set includes reflections between 35 and 1.

The TRTGn motif stabilizes the transcription initiation open complex.

The effect on transcription initiation by the extended -10 motif (5'-TRTG(n)-3'), positioned upstream of the -10 region, was investigated using a series of base substitution mutations in the alpha-amylase promoter (amyP). The extended -10 motif, previously referred to as the -16 region, is found frequently in Gram-positive bacterial promoters and several extended -10 promoters from Escherichia coli. The inhibitory effects of the non-productive promoter site (amyP2), which overlaps the upstream region of amyP, were eliminated by mutagenesis of the -35 region and the TRTG motif of amyP2.

Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C.

The enzymatic transformation of 2,4,6-trinitrotoluene (TNT) by purified XenB, an NADPH-dependent flavoprotein oxidoreductase from Pseudomonas fluorescens I-C, was evaluated by using natural abundance and [U-(14)C]TNT preparations. XenB catalyzed the reduction of TNT either by hydride addition to the aromatic ring or by nitro group reduction, with the accumulation of various tautomers of the protonated dihydride-Meisenheimer complex of TNT, 2-hydroxylamino-4,6-dinitrotoluene, and 4-hydroxylamino-2, 6-dinitrotoluene. Subsequent reactions of these metabolites were nonenzymatic and resulted in predominant formation of at least three dimers with an anionic m/z of 376 as determined by negative-mode electrospray ionization mass spectrometry and the release of approximately 0.

Cloning and sequence analysis of two Pseudomonas flavoprotein xenobiotic reductases.

The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from Pseudomonas putida II-B and P. fluorescens I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The P.

Transcriptional activation of the Bacillus subtilis ackA gene requires sequences upstream of the promoter.

Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B. subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene.

NADP, corepressor for the Bacillus catabolite control protein CcpA.

Expression of the alpha-amylase gene (amyE) of Bacillus subtilis is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in Bacillus and other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the amyE operator, amyO. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription.

The -16 region of Bacillus subtilis and other gram-positive bacterial promoters.

The Bacillus subtilis alpha-amylase promoter amy P contains an essential TGTG motif (-16 region) upstream of the -10 region. Mutations of this region significantly reduced in vitro promoter strength. A -15 G-->C transversion eliminated transcription from amy P by both B.

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species.

Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C. While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.

Contacts between Bacillus subtilis catabolite regulatory protein CcpA and amyO target site.

Catabolite control protein A (CcpA) is a global regulatory protein involved in catabolite repression and glucose activation in Gram-positive bacteria. cis -Acting DNA sequences, catabolite response elements ( cre s), involved in this regulatory system contain a 14 base pair (bp) region of dyad symmetry. CcpA, a repressor of the Lac I family, has been shown to bind specifically to cre s.

Significance of HPr in catabolite repression of alpha-amylase.

CcpA and HPr are presently the only two proteins implicated in Bacillus subtilis global carbon source catabolite repression, and the ptsH1 mutation in the gene for the HPr protein was reported to relieve catabolite repression of several genes. However, alpha-amylase synthesis by B. subtilis SA003 containing the ptsH1 mutation was repressed by glucose.

Specificity of DNA binding activity of the Bacillus subtilis catabolite control protein CcpA.

CcpA was purified from Escherichia coli BL21 (lambda DE3)/pLysS carrying plasmid pTSC5, which was constructed by inserting the ccpA gene into the polycloning site of pGEM4. The purified protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mass of 38 kDa but was eluted from a calibrated Bio-Gel P-100 column with an apparent mass of 75 kDa. Western blot (immunoblot) analysis revealed the presence of CcpA in E.

The -16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli.

The promoter (amyP) of the Bacillus subtilis alpha-amylase gene, which is recognized by E sigma A, has a three out of six match to the consensus promoter in both the -35 and -10 hexamers. Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions -18 to -15 (the -16 region) caused a 5-94-fold decrease in alpha-amylase production.

Characterization of a new sporulation factor in Bacillus subtilis.

We report the existence and partial purification of sporulation factor, which stimulates sporulation of Bacillus subtilis at low cell density. Proline or arginine is required for stimulation under the conditions of our assay. Sporulation factor is a small heat-stable substance produced by the cells during exponential growth phase.

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality.

Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors.

Expression of the alpha-amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidly metabolizable carbon sources. Glucose-mediated repression has previously been shown to involve a site near the transcriptional start-point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression.

Bacillus subtilis mutants with alterations in ribosomal protein S4.

Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsD1 and rpsD2, were located between the argGH and aroG genes, at 263 degrees on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 12 degrees.

Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis.

Catabolite repression of the Bacillus subtilis alpha-amylase gene (amyE) involves an operator sequence located just downstream of the promoter (amyR), overlapping the transcription start site. Oligonucleotide site-directed mutagenesis of this sequence identified bases required for catabolite repression. Two mutations increased both the 2-fold symmetry of the operator and the repression ratio.

Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene.

The amyR2 allele of the Bacillus subtilis alpha-amylase cis-regulatory region enhances production of amylase and transcription of amyE, the structural gene, by two- to threefold over amyR1. The amylase gene bearing each of these alleles was cloned on plasmids of about 10 to 15 copies per chromosome. Transcription of the cloned amylase gene by each amyR allele was activated at the end of exponential growth and was subject to catabolite repression by glucose.

Bacillus licheniformis alpha-amylase gene, amyL, is subject to promoter-independent catabolite repression in Bacillus subtilis.

Expression of the Bacillus licheniformis alpha-amylase gene, amyL, was temporally activated and subject to catabolite repression both in its natural host and when cloned on a 3.55-kilobase fragment in Bacillus subtilis. A subclone from which the promoter region of amyL and sequences upstream from the promoter were deleted had a low level of amylase activity.

Catabolite repression-resistant mutations of the Bacillus subtilis alpha-amylase promoter affect transcription levels and are in an operator-like sequence.

The amyR1 locus controls the regulated transcription of amyE, the structural gene encoding alpha-amylase in Bacillus subtilis. Transcription of amyE is activated in early stationary phase cells, and can be repressed by rapidly metabolized carbon sources such as glucose. Transcription of amyE initiates in vitro from a promoter recognized by the major vegetative form of RNA polymerase, E sigma 43.

Effect of decoyinine on the regulation of alpha-amylase synthesis in Bacillus subtilis.

Decoyinine, an inhibitor of GMP synthetase, allows sporulation in Bacillus subtilis to initiate and proceed under otherwise catabolite-repressing conditions. The effect of decoyinine on alpha-amylase synthesis in B. subtilis, an event which exhibits regulatory features resembling sporulation initiation, was examined.