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PubMed1994

Frequency modulation versus amplitude modulation discrimination: evidence for a second frequency modulation encoding mechanism.

The encoding mechanisms for amplitude modulation (AM) and frequency modulation (FM) were investigated using AM-FM discrimination tasks. In the first experiment, AM and FM were set at equally detectable levels within a trial, and discrimination thresholds were obtained adaptively in a 3IFC task. Here, AM-FM discrimination thresholds were considerably larger than both AM and FM detection thresholds. This is consistent with an encoding system whereby AM and FM are partially encoded by the same mechanism. In the second experiment, performance on AM-FM discrimination is measured with a fixed-level procedure. Psychometric functions obtained for a constant modulation depth of AM were nonmonotonic with FMs modulation index beta and each displayed a single minimum. The nonmonotonic nature of the functions is consistent with a model in which FM is encoded primarily with the same mechanism that encodes AM but also with a second mechanism, probably related to changes in instantaneous frequency, that is independent of the mechanism that extracts AM. The fact that minima in the discrimination psychometric functions increase from d' = 0 as beta increases indicates that the information encoded by the second mechanism becomes more detectable with increasing beta.

Auditory Perception↗
PubMed1992

Transposition of the gene encoding a TEM-12 extended-spectrum beta-lactamase.

An isolate of Klebsiella oxytoca from the blood culture of a child with leukemia was found to produce two beta-lactamases, at least one of which conferred resistance to ceftazidime. Genes encoding both enzymes were located on a single self-transmissible 100-kb plasmid, pOZ201. This plasmid was introduced into Escherichia coli UB5201 (pACYC184), and the gene encoding one beta-lactamase was transposed onto plasmid pACYC184 by exploiting a gene dosage effect. The transposable gene was found to encode a TEM-12 enzyme as determined by nucleotide sequencing. This gene was subsequently transposed onto plasmid pUB307. The transposable element encoding the TEM-12 enzyme has been designated Tn841. Both plasmids pACYC184::Tn841 and pUB307::Tn841 were shown to encode a beta-lactamase with the same isoelectric point and substrate profile as the TEM-12 beta-lactamase. Transposon Tn841, at approximately 7 kb, is larger than TnA (4.8 kb) and transposes at a lower frequency. Although it produced a resolvase which can complement the resolvase of Tn3, its transposase function was not able to complement the transposition of a TnA element which lacked transposase. The occurrence of a gene encoding an extended-spectrum beta-lactamase on a transposable element in a clinically significant bacterium is potentially a cause for concern for the spread of resistance to the extended-spectrum cephalosporins.

Base Sequence↗
PubMed1992

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.

Amino Acid Sequence↗
PubMed1998

Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis.

Isolated outer membranes of Borrelia burgdorferi were used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [3H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) of B. burgdorferi N40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467-3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of the dbp gene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 and dbp comprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts of B. burgdorferi isolated outer membranes. Mice infected with B. burgdorferi produced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbp operon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.

Adhesins, Bacterial↗
PubMed2004

Diversity and host range of Shiga toxin-encoding phage.

Shiga toxin 2 (Stx2) from the foodborne pathogen Escherichia coli O157:H7 is encoded on a temperate bacteriophage. Toxin-encoding phages from C600::933W and from six clinical E. coli O157:H7 isolates were characterized for PCR polymorphisms, phage morphology, toxin production, and lytic and lysogenic infection profiles on O157 and non-O157 serotype E. coli. The phages were found to be highly variable, and even phages isolated from strains with identical pulsed-field gel electrophoresis profiles differed. Examination of cross-plaquing and lysogeny profiles further substantiated that each phage is distinct; reciprocal patterns of susceptibility and resistance were not observed and it was not possible to define immunity groups. The interaction between Shiga toxin-encoding phage and intestinal E. coli was examined. Lytic infection was assessed by examining Shiga toxin production following overnight incubation with phage. While not common, lytic infection was observed, with a more-than-1,000-fold increase in Stx2 seen in one case, demonstrating that commensal E. coli cells can amplify Shiga toxin if they are susceptible to infection by the Shiga toxin-encoding phages. Antibiotic-resistant derivatives of the Stx2-encoding phages were used to examine lysogeny. Different phages were found to lysogenize different strains of intestinal E. coli. Lysogeny was found to occur more commonly than lytic infection. The presence of a diverse population of Shiga toxin-encoding phages may increase the pathogenic fitness of E. coli O157:H7.

Bacteriophages↗
PubMed1988

Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae.

We have cloned and expressed in Escherichia coli a gene encoding a 15,000-apparent-molecular-weight peptidoglycan-associated outer membrane lipoprotein (PAL) of Haemophilus influenzae. The nucleotide sequence of this gene encodes an open reading frame of 153 codons with a predicted mature protein of 134 amino acids. The amino acid composition and sequence of the predicted mature protein agree with the chemically determined composition and partial amino acid sequence of PAL purified from H. influenzae outer membranes. We have also identified a second gene from H. influenzae that encodes a second 15,000-apparent-molecular-weight protein which is recognized by antiserum against PAL. This protein has been shown to be a lipoprotein. The nucleotide sequence of this gene encodes an open reading frame of 154 codons with a predicted mature protein of 136 amino acids and has limited sequence homology with that of the gene encoding PAL. Southern hybridization analysis indicates that both genes exist as single copies in H. influenzae chromosomal DNA. Both genes encode polypeptides which have amino-terminal sequences similar to those of reported membrane signal peptides and are associated primarily with the outer membrane when expressed in E. coli.

Amino Acid Sequence↗
PubMed1991

The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system.

Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes. A second site mutation in malI abolishes the constitutive expression. The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J. Reidl, K. Römisch, M. Ehrmann, and W. Boos, J. Bacteriol. 171:4888-4899, 1989). We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY. MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE). When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose. Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX. MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion. We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system. The second gene in the operon, malY, encodes a protein of 43,500 daltons. Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences. The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant. The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose. This is not the case in a malT(Con) strain that expresses the mal genes constitutively. We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis.

Amino Acid Sequence↗
PubMed1992

Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the Asd+ vector pYA292, and the construct was introduced into the avirulent delta cya delta crp delta asd S. typhimurium chi 3987 for oral immunization of birds. The gene encoding the 21-kDa protein was expressed equivalently in B. avium 197, delta asd E. coli chi 6097, and S. typhimurium chi 3987 and was localized primarily in the cytoplasmic membrane and outer membrane. In preliminary studies on oral inoculation of turkey poults with S. typhimurium chi 3987 expressing the gene encoding the B. avium 21-kDa protein, it was determined that a single dose of the recombinant Salmonella vaccine failed to elicit serum antibodies against the 21-kDa protein and challenge with wild-type B. avium 197 resulted in colonization of the trachea and thymus with B. avium 197.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of the adi gene, which encodes the biodegradative acid-induced arginine decarboxylase of Escherichia coli.

Arginine decarboxylase (encoded by adi) is induced under conditions of acidic pH, anaerobiosis, and rich medium. The DNA sequence of a 3-kb fragment of the Escherichia coli chromosome encoding biodegradative arginine decarboxylase was determined. This sequence encodes a protein of 755 amino acids with a molecular size of 84,420 daltons. The molecular weight and predicted Adi amino acid composition agree with those found in earlier work. The amino acid sequence of arginine decarboxylase showed homology to those of three other decarboxylases of E. coli: (i) CadA, encoding lysine decarboxylase; (ii) SpeC, encoding biosynthetic ornithine decarboxylase; and (iii) SpeF, encoding biodegradative ornithine decarboxylase and the lysine decarboxylase of Hafnia alvei. Unlike SpeC and SpeF, Adi is not similar to the biosynthetic arginine decarboxylase, SpeA. adi is also dissimilar to cadA and speF in that it does not appear to be part of an operon containing a metabolically related transport protein, indicating that it represents a new type of biodegradative decarboxylase regulation. Transcriptional fusions between fragments upstream of adi and lacZ, primer extension, and site-directed mutagenesis experiments defined the pH-regulated promoter. Deletion analysis of the upstream region and cloning of fragments to make adi::lacZ protein fusion implicated a region beyond an upstream SspI site in pH regulation. Induction of adi in the presence of sublethal concentrations of novobiocin or coumermycin A1, inhibitors of DNA gyrase, was dramatically decreased, indicating that DNA supercoiling is involved in adi expression. These results and those of promoter structure studies indicated that acid regulation of adi may involve a mechanism different from that of acid regulation of cad.

Amino Acid Sequence↗
PubMed1994

Growth phase-dependent transcription of the genes that encode the two methyl coenzyme M reductase isoenzymes and N5-methyltetrahydromethanopterin:coenzyme M methyltransferase in Methanobacterium thermoautotrophicum delta H.

The genes encoding the two isoenzymes of methyl coenzyme M reductase (MRI and MRII) in Methanobacterium thermoautotrophicum delta H have been cloned and sequenced. The MRI-encoding mcr operon (mcrBDCGA) has been located immediately upstream from the mtr operon (mtrEDCBA) that encodes N5-methyltetrahydromethanopterin:coenzyme M methyltransferase, the enzyme that catalyzes the step preceding the MR-catalyzed reaction in methanogenesis. The MRII-encoding mrt operon (mrtBDGA) has been located between the operon that encodes the methyl viologen-reducing hydrogenase and an open reading frame (designated pyrC) predicted to encode dihydroorotase. Surprisingly, the mrt operon has been found to contain only four genes (mrtBDGA), lacking the equivalent of the mcrC gene that is present in all mcr operons. A protocol that isolates transcripts intact from M. thermoautotrophicum delta H cells has been developed and used, with primer extension and Northern (RNA) blot procedures, to identify the sites of transcription initiation upstream of the mcr, mrt, and mtr operons and to determine the relative numbers of these transcripts in cells at different growth stages. Transcription of the mrt operon was found to occur only at early times in batch cultures and was then replaced by transcription of the mcr operon. Transcripts of the mtr operon were detectable at all times; however, at early times, all mtr transcripts were initiated at the mtr promoter, whereas at later times, during mcr transcription, approximately 3% of mcr transcripts were extended to generate mcr plus mtr transcripts that constituted approximately 20% of all mtr transcripts present.

Amino Acid Sequence↗
PubMed1994

Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase.

Expression of sdhCDAB (encoding succinate dehydrogenase) and lctD (encoding the flavin-linked L-lactate dehydrogenase) is elevated aerobically and repressed anaerobically in Escherichia coli. The repression is initiated by autophosphorylation of the sensor protein ArcB, followed by phosphoryl group transfer to the regulator ArcA. ArcA-P, a global transcriptional regulator, then prevents sdh and lct expression. The stimulus for ArcB is not O2 deficiency per se. In vitro experiments showed that ArcB phosphorylation is enhanced by pyruvate, D-lactate, acetate, and NADH, the concentrations of which are likely to increase with the lack of an effective exogenous electron sink. In addition to their aerobic function, the two primary dehydrogenases also have roles in anaerobic nitrate respiration. Results presented here indicate that the increase of sdh and lct expression by nitrate depended on its chemical reduction, which in turn diminished the ArcA-P pool. Unexpectedly, a mutation in the fnr gene (encoding a global regulator involved in anaerobic metabolism) also alleviated the anaerobic repressions. Mutations in arcB or arcA were epistatic over that of fnr. Moreover, since this relief was counteracted by pyruvate in the growth medium, Fnr appears to affect formation of stimuli for ArcB. It is possible that Fnr also indirectly affects some of the other members of the arcA modulon, e.g., cyoABCDE (encoding the cytochrome o complex), cydAB (encoding the cytochrome d complex), and sodA (encoding the manganese-dependent superoxide dismutase).

Anaerobiosis↗
PubMed1995

Biochemical characterization of gapB-encoded erythrose 4-phosphate dehydrogenase of Escherichia coli K-12 and its possible role in pyridoxal 5'-phosphate biosynthesis.

One step in de novo pyridoxine (vitamin B6) and pyridoxal 5'-phosphate biosynthesis was predicted to be an oxidation catalyzed by an unidentified D-erythrose-4-phosphate dehydrogenase (E4PDH). To help identify this E4PDH, we purified the Escherichia coli K-12 gapA- and gapB-encoded dehydrogenases to homogeneity and tested whether either uses D-erythrose-4-phosphate (E4P) as a substrate. gapA (gap1) encodes the major D-glyceraldehyde-3-phosphate dehydrogenase (GA3PDH). The function of gapB (gap2) is unknown, although it was suggested that gapB encodes a second form of GA3PDH or is a cryptic gene. We found that the gapB-encoded enzyme is indeed an E4PDH and not a second GA3PDH, whereas gapA-encoded GA3PDH used E4P poorly, if at all, as a substrate under the in vitro reaction conditions used in this study. The amino terminus of purified E4PDH matched the sequence predicted from the gapB DNA sequence. Purified E4PDH was a heat-stable tetramer with a native molecular mass of 132 kDa. E4PDH had an apparent Km value for E4P [Kmapp(E4P)] of 0.96 mM, an apparent kcat catalytic constant for E4P [kcatapp(E4P)] of 200 s-1, Kmapp(NAD+) of 0.074 mM, and kcatapp(NAD+) of 169 s-1 in steady-state reactions in which NADH formation was determined. From specific activities in crude extracts, we estimated that there are at least 940 E4PDH tetramer molecules per bacterium growing in minimal salts medium plus glucose at 37 degrees C. Thin-layer chromatography confirmed that the product of the E4PDH reaction was likely the aldonic acid 4-phosphoerythronate. To establish a possible role of E4PDH in pyridoxal 5'-phosphate biosynthesis, we showed that 4-phosphoerythronate is a likely substrate for the 2-hydroxy-acid dehydrogenase encoded by the pdxB gene. Implications of these findings in the evolution of GA3PDHs are also discussed. On the basis of these results, we propose renaming gapB as epd (for D-erythrose-4-phosphate dehydrogenase).

Alcohol Oxidoreductases↗
PubMed1996

Induction of the gap-pgk operon encoding glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase of Xanthobacter flavus requires the LysR-type transcriptional activator CbbR.

In a previous study, a gene (pgk) encoding phosphoglycerate kinase was isolated from a genomic library of Xanthobacter flavus. Although this gene is essential for autotrophic growth, it is not located within the cbb operon encoding other Calvin cycle enzymes. An analysis of the nucleotide sequence upstream from pgk showed the presence of a gene encoding glyceraldehyde-3-phosphate dehydrogenase and the 3' end of an open reading frame encoding a protein which is 50% identical to transketolase encoded by cbbT of X. flavus. Gene fusions between pgk and lacZ demonstrated that the gap and pgk genes are organized in an operon. Induction of the Calvin cycle in heterotrophically growing cells resulted in a sixfold increase in phosphoglycerate kinase activity in parallel with the appearance of ribulosebisphosphate carboxylase activity. This superinduction of phosphoglycerate kinase did not occur in an X. flavus strain in which cbbR, encoding the transcriptional activator of the cbb operon, was disrupted. The failure to superinduce the gap-pgk operon is not caused by the absence of a functional Calvin cycle, since the expression of this operon in an X. flavus strain with a defective ribulosebisphosphate carboxylase enzyme was the same as the expression in the wild type. It is therefore concluded that the expression of both the cbb and gap-pgk operons is controlled by CbbR.

Amino Acid Sequence↗
PubMed1997

Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon.

Adenosine Triphosphate↗
PubMed1997

Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1.

The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bac1, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bac1 ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bac1, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broad-spectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.

Amino Acid Sequence↗
PubMed1998

Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12.

We have identified, cloned, and sequenced the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in Escherichia coli K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (hcaA1A2CBD) and two additional genes transcribed in the opposite direction that encode a potential permease (hcaT) and a regulator (hcaR). Sequence comparisons revealed that while hcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E. coli. The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2, 3-dihydroxy derivative. Further catabolism of DHPP requires the mhp-encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. García, and E. Díaz, J. Bacteriol. 179:2573-2581, 1997). Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent mhp- and hca-encoded pathways are also functional in S. typhimurium, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.

Amino Acid Sequence↗
PubMed2000

The Pseudomonas aeruginosa devB/SOL homolog, pgl, is a member of the hex regulon and encodes 6-phosphogluconolactonase.

A cyclic version of the Entner-Doudoroff pathway is used by Pseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form the hex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOL family of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5' end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgl and propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.

Aldehyde-Lyases↗
PubMed2000

The Saccharomyces cerevisiae ICL2 gene encodes a mitochondrial 2-methylisocitrate lyase involved in propionyl-coenzyme A metabolism.

The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologous ICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect of icl1 null mutants. It has therefore been suggested that ICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whether ICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenic icl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 and icl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2 mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that the ICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction of ICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.

Acyl Coenzyme A↗