Characterization of squid crystallin genes. Comparison with mammalian glutathione S-transferase genes.

Previous experiments have indicated that the crystallins of the squid lens (S-crystallins) are evolutionarily related to glutathione S-transferases (GST) (EC 2.5.1.18). Here we confirm by peptide sequencing that the crystallins of the lens of the squid Ommastrephes sloani pacificus comprise a family of GST-like proteins. Squid lens extracts showed 400 times less GST activity than those of liver using 1-chloro-2,4-dinitrobenzene as a substrate, suggesting that the abundant GST-like crystallins lack enzymatic activity. Four different cDNAs (pSL20-1, pSL18, pSL11, and pSL4) showed 20-25% similarity in homologous regions with mammalian GST polypeptides. pSL20-1, pSL18, and pSL4 each encode an S-crystallin with a unique internal peptide that is unrelated to mammalian GSTs or any other sequence in GenBank. The S-crystallin family is encoded in a minimum of 9-10 genes, and the exon-intron structures of at least two of these (SL20-1 and SL11) are similar to those of the mammalian GST genes. The SL20-1 gene has six exons, with the its unique internal peptide encoded precisely in exon 4; the SL11 gene lacks a unique internal peptide and has five exons. Experiments using bacterial chloramphenicol acetyltransferase as a reporter gene showed that at least 84 and 111 base pairs of 5'-flanking sequence are needed for function of the SL20-1 and SL11 promoters, respectively, in a transfected rabbit lens epithelial cell line (N/N1003A). Within these regions each has a putative TATA box and an upstream AP-1 site overlapping with antioxidant responsive-like elements, which are regulatory elements in the rat GST Ya and quinone reductase genes responsive to oxidative stress.     

Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1

The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1. Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli. The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S. lividans 66 was determined. All except one of the inserts in the 1.52-kb essential region inactivated replication. A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment. The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.     

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.     

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway.

The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.     

Cloning and expression in Escherichia coli of a gene encoding proline oxidase of Serratia marcescens.

Proline plays a central role in the biosynthesis of prodigiosin by Serratia marcescens. Proline catabolism takes place by oxidation catalysed by the enzyme proline oxidase encoded by the gene putA. A gene bank of chromosomal DNA from S. marcescens was constructed using the plasmid vector pBR328, and then recombinant DNA was used in transformation experiments with Escherichia coli HB 101 as recipient strain. One of the recombinant plasmids, pSL001, was encoded for proline oxidase. Subcloning experiments led to a second plasmid pSL008 able to maintain proline oxidase activity.     

pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo . Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli . The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.     

Expression of Pseudomonas fluorescens D-galactose dehydrogenase in E. coli

To investigate the heterologous expression of Pseudomonas genes in Escherichia coli we have cloned P. fluorescens DNA in an E. coli [cosmid] system. A colony bank representing the whole P. fluorescens chromosome was screened immunologically using a modification of the method described by Broome and Gilbert (1978). Radioactive labelling of the antibodies was replaced by conjugation with horseradish peroxidase. Among 523 E. coli colonies one was D-galactose dehydrogenase-positive. The expression of this enzyme in primary clones was lower than in the uninduced Pseudomonas. Subcloning of the D-galactose dehydrogenase gene, in vitro mutagenesis of the DNA, and coupling to a strong E. coli promoter yielded an E. coli strain that produces 90 times more of the enzyme than the induced P. fluorescens.     

Evidence for the cloning of Deinococcus radiodurans DNA fragments that render Escherichia coli radiation resistant

DNA from the radiation-resistant bacterium Deinococcus radiodurans was isolated and used to generate a cosmid library. This cosmid library was grown in Escherichia coli and radiation-resistant E. coli were isolated. Following exposure to 1000 Gy the radiation-resistant transformants exhibited a survival of approximately 10(-1) instead of the 10(-11) exhibited by the nontransformed E. coli. Smaller fragments of DNA were subcloned from the radiation-resistant E. coli; these fragments bestow similar levels of radiation resistance (ratio of slopes = 6.8) to native E. coli upon transfection.     

A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase

Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhEMeta. The AdhEMeta protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhEMeta did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhEMeta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhEMeta is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhEMeta did, however, confer one phenotype to E. coli. AdhE- mutants of E. coli fail to ferment glucose and introduction of adhEMeta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions.     

A recB recC sbcB recJ host prevents recA-independent deletions in recombinant cosmid DNA propagated in Escherichia coli.

Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.     

Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.     

Suppression of an Escherichia coli secAts mutant by a gene cloned from Staphylococcus carnosus

Escherichia coli mutant MM52 (secA(ts)) was transformed with a cosmid library from Staphylococcus carnosus, and a recombinant cosmid (pBO23) allowing growth at the non-permissive temperature (42 degrees C) was isolated. pBO23 also restored the growth defects of E. coli mutants IQ85 (secY(ts)) and IT41 (lep(ts)). Nucleotide sequencing revealed that the DNA fragment responsible for the suppression effect codes for a S. carnosus protein highly homologous to the ribosomal protein L13 of E. coli. The staphylococcal L13 protein was efficiently incorporated into E. coli ribosomes. Possible explanations for the effect of this polypeptide on the growth of temperature-sensitive E. coli secretion mutants are discussed.     

Identification of active site residues in Bradyrhizobium japonicum acetyl-CoA synthetase.

Acetyl-CoA synthetase (ACS) catalyses the activation of acetate to acetyl-CoA in the presence of ATP and CoA. The gene encoding Bradyrhyzobium japonicum ACS has been cloned, sequenced, and expressed in Escherichia coli. The enzyme comprises 648 amino acid residues with a calculated molecular mass of 71,996 Da. The recombinant enzyme was also purified from the transformed E. coli. The enzyme was essentially indistinguishable from the ACS of B. japonicum bacteroids as to the criteria of polyacrylamide gel electrophoresis and biochemical properties. Based on the results of database analysis, Gly-263, Gly-266, Lys-269, and Glu-414 were selected for site-directed mutagenesis in order to identify amino acid residues essential for substrate binding and/or catalysis. Four different mutant enzymes (G263I, G266I, K269G, and E414Q) were prepared and then subjected to steady-state kinetic studies. The kinetic data obtained for the mutants suggest that Gly-266 and Lys-269 participate in the formation of acetyl-AMP, whereas Glu-414 may play a role in acetate binding.     

Growth of Salmonella typhimurium SL5319 and Escherichia coli F-18 in mouse cecal mucus: role of peptides and iron

Abstract Escherichia coli F-18, a normal human fecal isolate, and Salmonella typhimurium SL5319, an avirulent strain, are known to colonize the streptomycin-treated CD-1 mouse large intestine by utilizing nutrients present in intestinal mucus for growth. Moreover, previous experiments suggested the possibility that E. coli F-18 and S. typhimurium SL5319 utilized different mucus nutrients. Therefore, mouse cecal mucus was fractionated into high and low molecular weight components, and each fraction was inoculated either simultaneously or separately with E. coli F-18 and S. typhimurium SL5319. A 50 kd fraction was found in which the growth of S. typhimurium SL5319 suppressed growth of E. coli F-18. Evidence is presented that in this fraction S. typhimurium SL5319 utilizes peptides, presumably generated by mucus proteases, as a source of amino acids for growth. Furthermore, it is shown that S. typhimurium SL5319 grows in this 50 kd fraction with a generation time of 27 min in the presence of at most 7 μg of carbohydrate per ml and 2.2 μg of peptide per ml, and that S. typhimurium SL5319 suppresses E. coli F-18 growth in this fraction by sequestering iron. The data are discussed with respect to the role of peptide utilization and iron sequestration in the ability of S. typhimurium SL5319 to colonize the mouse large intestine.     

Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 for expression of the vibrio neuraminidase (NANase) gene nanH (N-acylneuraminate glycohydrolase). Positive clones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after screening 683 cosmid isolates with a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and was located within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was the NANase structural gene was obtained by transposon mutagenesis and by purification and comparison of the cloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of the first 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined by automated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotide sequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that was apparently processed from NANase in both V. cholerae and E. coli. In contrast to V. cholerae, E. coli nanH+ clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmic compartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion were temporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics were observed in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might be controlled by some of the same signals as in the parent V. cholerae strain.     

Overexpression of salicylate hydroxylase and the crucial role of lys(163) as its NADH binding site

Expression systems for the sal gene encoding salicylate hydroxylase from Pseudomonas putida S-1 were examined and some constructs were expressed in these systems. By cultivation of Escherichia coli BL21 (DE3)/pSAH8 in LB medium at 37 degrees C with isopropyl-b-D-thiogalactopyranoside as the inducer, salicylate hydroxylase was overexpressed mainly in the form of inclusion bodies. Lower temperature cultivation at 20 degrees C after induction resulted in a large amount of the enzyme in the soluble form. The E. coli clone harboring the recombinant plasmid produced a 45 kDa protein that appeared to be electrophoretically and immunochemically identical to the P. putida enzyme and contained the same N-terminal amino acid sequence. This recombinant DNA product also exhibited properties characteristic of a flavoprotein and was fully functional as salicylate hydroxylase. Based on chemical modification of the salicylate hydroxylase from P. putida, Lys163 was previously proposed to be the NADH binding site. In this study, to obtain a better understanding of the predicted role of Lys163, this residue in the active center of salicylate hydroxylase was replaced with Arg, Gly, or Glu by conventional site-directed mutagenesis. Kinetic studies using these mutant enzymes and the recombinant enzyme revealed increases in apparent K(m) values for NADH in the order of wild-type enzyme > K163R > K163G > K163E, with some decreases in V(max). Examination of the recombinant enzyme and K163G indicated that the pH dependency of K(m) on NADH with pK(a) 10.5 is lost by mutation despite the lack of changes in V(max) values, suggesting a requirement for the lysine residue as the NADH binding site. Based on these results, Lys163 is proposed to play a role in the binding of NADH at the active site through an ionic bond rather than playing a role in catalysis.     

Evolution of enzymatic activity in the enolase superfamily: functional studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis

o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme first was identified as an N-acylamino acid racemase (NAAAR), with the optimal substrates being the enantiomers of N-acetyl methionine. This laboratory subsequently discovered that this protein is a much better catalyst of the OSBS reaction, with the value of k(cat)/K(M), for dehydration, 2.5 x 10(5) M(-1) s(-1), greatly exceeding that for 1,1-proton transfer using the enantiomers of N-acetylmethionine as substrate, 3.1 x 10(2) M(-1) s(-1) [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-8]. The efficiency of the promiscuous NAAAR reaction is enhanced with alternate substrates whose structures mimic that of the SHCHC substrate for the OSBS reaction, for example, the value of k(cat)/K(M) for the enantiomers of N-succinyl phenylglycine, 2.0 x 10(5) M(-1) s(-1), is comparable to that for the OSBS reaction. The mechanisms of the NAAAR and OSBS reactions have been explored using mutants of Lys 163 and Lys 263 (K163A/R/S and K263A/R/S), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSBS from Escherichia coli. Although none of the mutants display detectable OSBS or NAAAR activities, K163R and K163S catalyze stereospecific exchange of the alpha-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and K263R and K263 catalyze the stereospecific exchange the alpha-hydrogen of N-succinyl-(R)-phenylglycine, consistent with formation of a Mn2+-stabilized enolate anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for racemization. That this enzyme can catalyze two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity.     

Cloning of an insecticidal toxin gene of Bacillus thuringiensis subsp. tenebrionis and its expression in Escherichia coli cells

Abstract A structural gene of a crystal protein toxic for coleoptera larvae was cloned from plasmid DNA of Bacillus thuringiensis subsp. tenebrionis (BTT). The DNA was partially digested with restriction enzyme Bam HI and fragments were inserted into cosmid pHC79. In Western blot analysis extracts from infected Escherichia coli cells revealed expression of the BTT crystal protein in antibiotic-resistant cells. Cell lysates from a selected E. coli clone were toxic for larvae of the Colorado potato beetle ( Leptinotarsa decemlineata ). The electrophoretic mobility in SDS gels of crystal protein from E. coli cells was 68 kDa and 74 kDa as observed for BTT-toxin in B. thuringiensis extracts. The cosmids obtained were unstable during cellular propagation. The deletion product still carried the δ-endotoxin gene.     

RecA-independent high-frequency deletion of recombinant cosmid DNA in Escherichia coli

Segments of DNA were deleted from recombinant cosmid DNAs during propagation in Escherichia coli hosts in liquid culture. DNAs of more than 1000 cosmids propagated in various E. coli hosts were analysed by agarose gel electrophoresis (AGE). The effects of vectors, insert DNAs and host genetic characters on the formation of deletions were examined. The probability of deletion and the pattern of deletion bands observed by AGE differed from clone to clone, and after extensive culture the deletion band patterns remained almost constant during further culture. Most recombinant clones eventually showed deletion during prolonged liquid culture. Mutations in the recA gene of E. coli hosts, including a deletion mutation, did not prevent deletion. Most deletions occurred in the insert portions of cosmid DNAs. Nucleotide sequence analysis of six deletion junctions in test cosmid cMB15 demonstrated that deletions occurred between two short complete direct repeats of about 4-10 bp, irrespective of whether the cosmid was propagated in a recA host or a rec+ host. Some deletions occurred at the same sites either in a recA host or a rec+ host. These results suggest that the deletion events are mainly mediated by a recA-independent recombination system(s) of E. coli host cells.