The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.     

N-carbobenzyloxy-D-aspartic acid as a competitive inhibitor of indole-3-acetyl-L-aspartic acid hydrolase of Enterobacter agglomerans

The gene for a specific IAA-asp hydrolase from Enterobacteragglomerans, iaaspH, is a potentially useful tool for modificationofIAA homeostasis in higher plants that use the IAA-asp oxidation pathway forauxin catabolism. In order to optimize the utility of this gene for plantmodification and to increase the success of obtainingiaaspH transformed plants from culture, we haveinvestigated aspects of IAA-asp hydrolase catalysis. The catalyticcharacteristics of the IAA-asp hydrolase from Enterobacteragglomerans was studied using ten compounds that are structuralanalogues of IAA-asp. These compounds were tested as potential IAA-asphydrolasesubstrates as well as for inhibition of IAA-asp hydrolysis. Among them,N-carbobenzyloxy-D-aspartic acid (N-CBZ-D-asp) and N-CBZ-L-asp were found to bethe strongest inhibitors with more than 80% inhibition of IAA-asp hydrolysis.Aspartyl-L-aspartic acid and a asp-ser-asp-pro-arg peptide also showed stronginhibitory activities, reducing rates of IAA-L-asp hydrolysis, when added atequal molar amounts relative to the substrate, by 60% and 65%, respectively.N-CBZ-D-asp was chosen for further kinetic studies and for studies of itstoxicity in relation to seed germination because it was a strong inhibitor,exhibited a very low rate of hydrolysis by the IAA-asp hydrolase and wascommercially available. N-CBZ-D-asp was shown to be a competitive inhibitor forthe Enterobacter agglomerans IAA-asp hydrolase with aK _i value of 1.22 mM. Studies oftomato seed germination showed that N-CBZ-D-asp did not affect the rate of seedgermination at up to 1 mM, but the growth rate of seedlings wassignificantly reduced when the concentration in the medium was 0.5mM and higher. These results indicate that, at suitableconcentrations, N-CBZ-D-asp should be a useful tool for control of low levelexpression of the iaaspH in transgenic plants duringcritical stages of plant regeneration from culture.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998     

The cloning of Aspergillus nidulans mitochondrial DNA in Escherichia coli on plasmid pBR322

Summary We report the cloning of almost the entire mitochondrial DNA of Aspergillus nidulans on plasmid pBR322 in Escherichia coli. Only one fragment containing about 11% of the mitochondrial genome has not been cloned. We believe that this fragment cannot be maintained in E. coli on the pBR322 plasmid.     

AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

 Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf. Received: 30 November 1998 / Accepted: 21 April 1999     

Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a λgt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of M_r 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50 kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors. Received: 14 January 1998 / Accepted: 31 March 1998     

Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12

Genes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli. The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E. coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region). Transport studies in E. coli and B. stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B. stearothermophilus ATCC7953. Nucleotide sequence analysis of a 3.6 kb fragment of pAM 1750 revealed three open reading frames (ORFs). One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments. MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MaIG protein, a member of a binding protein-dependent transport system in E. coli. The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E. coli mutants. Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E. coli, the protein was very poorly expressed and could not be identified.     

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38 994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-l-alanine amidase, which has a M_r value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.     

Insertion specificity and trans -activation of IS801

The transposable element IS801, isolated from plasmid pMMC7105 of Pseudomonas syringae pv. phaseolicola, transposes in Escherichia coli to plasmid targets, expressing a relatively relaxed target specificity. The target sequences are tetramers with homology with the left terminus (GAAC) of the transposing unit, the alternative targets being GAAC, GGAC, CAAG, and CGAC. In the areas flanking IS801 in 13 different locations, no similarities other than the target tetramer were observed. The transposase is physically and functionally separable from the transposing unit since transposition of constructs carrying marker genes occurs with the transposase expressed in trans. The IS801 transposase shows amino acid sequence homology to the transposases of the E. coli elements IS91 and IS1294. These tranposases contain conserved amino acid motifs found in the replicases of certain plasmids that replicate as rolling circles. Received: 18 March 1998 / Accepted: 15 August 1998     

Peroxyl Radical Scavenging Activity of Caffeic Acid and Its Related Phenolic Compounds in Solution

The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.     

Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site

The integrase of the temperate bacteriophage mv4 catalyzes site-specific recombination between the phage attP site and the attB site of the host during lysogenization of Lactobacillus delbrueckii subsp. bulgaricus. The mv4 integrase also functions in a wide variety of gram-positive bacteria and in Escherichia coli. In this report, in vitro and in vivo recombination assays were developed and the integrase was purified in order to study in greater detail the mv4 attP × attB recombination event. In a cell-free extract of E. coli at 42° C, the mv4 integrase promotes efficient in vitro recombination between a supercoiled attP-containing plasmid and a linear attB fragment. The integrase, which was purified to apparent homogeneity, showed no absolute requirement for accessory factors, unlike the majority of the lambda Int family of recombinases. Deletion derivatives of the attP site were constructed and tested for recombination with the attB site in vitro. A 234-bp DNA fragment containing five scattered putative mv4 Int-binding sites was sufficient for function of the attP site. In contrast to the right arm of attP, most of the left arm could be deleted without drastically reducing the recombination efficiency. In vivo in E. coli, mv4 Int catalyzed recombination in trans between attP and attB sites present on two separate plasmids. This property was used to confirm in vivo the results of the deletion analysis of the attP site performed in vitro. Received: 22 July 1998 / Accepted: 4 June 1999     

Cloning of pEA3, a large plasmid ofEnterobacter agglomerans containing nitrogenase structural genes

Summary A clone bank of an indigenous plasmid ofEnterobacter agglomerans containing structural nitrogen-fixation (nif) genes was established in a non-mobilisable, multicopy derivative of the cosmid vector pHC79. The restriction enzyme Bam HI was used to establish the clone bank and it was found that 96% of the clones contained inserts. The clones containingnif-genes were identified by Southern hybridisation usingKlebsiella pneumoniae nif DNA (KpnifHDKY) as the radioactive probe. Thenif-genes ofE. agglomerans showed extensive homology to those ofK. pneumoniae but the restriction enzyme fragment patterns of thenif-genes ofE. agglomerans were different. The plasmid bornenif-genes ofE. agglomerans are clustered as inK. pneumoniae.     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).     

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

Summary A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27 513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M _r value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M _r value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.