Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.     

Comparative Study of Isoenzyme Formation of Bacterial β-Galactosidase

The enzyme beta-galactosidase was studied in crude extracts of Escherichia coli 3300, E. coli grown on a selenium and sulfur medium, Salmonella typhimurium F-lac, Serratia marcescens F-lac, S. marcescens P-lac, Proteus mirabilis F-lac, P. mirabilis P-lac, Aeromonas formicans, and Streptococcus lactis. The isoenzymes could be demonstrated by an alternative histochemical technique. Different isoenzyme patterns were found to be determined by the beta-galactosidase structural gene and not by the cytoplasm within which the beta-galactosidase was formed. In addition, the beta-galactosidases from strains which form isoenzymes were more stable to heat and urea treatments than the enzyme formed by those organisms which produce reduced amounts of, or no, isoenzyme.     

Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.     

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.     

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.     

Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media

This study compared the performance of LMX(R) broth (LMX), Chromocult Coliform(R) agar (CC) and Chromocult Coliform agar plus cefsulodin (10 microg ml-1) (CC-CFS), with standard methods multiple tube fermentation (MTF), for the enumeration of total coliforms and Escherichia coli from marine recreational waters. LMX and CC are two media designed to concurrently detect total coliform (TC) bacteria and E. coli by the specific action of beta-galactosidase (total coliforms) and beta-glucuronidase (E. coli). Overall results for the TC test showed that LMX, CC and MTF recovered 2.63, 1.95 and 1.90 times as many TCs as CC-CFS, respectively. Data from the multiple range test showed significant differences (P < 0.05) between TC counts on CC-CFS and LMX. The traditional MTF was less sensitive for E. coli enumeration. However, there was no statistically significant differences between LMX, CC, CC-CFS and the MTF method for E. coli enumeration. Background interference was reduced on CC-CFS and the counts obtained reflected more accurately the number of TCs. Therefore, the contribution of beta-galactosidase positive, non coliform bacteria (Aeromonas spp. and Vibrio spp.) to TC counts should not be neglected.     

Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.     

Enzyme polymorphism and genetic population structure in Escherichia coli and Shigella

Electrophoretically demonstrable variation in 12 enzymes was studied in more than 1 600 isolates of Escherichia coli from human and animal sources and in 123 strains of the four species of Shigella. All 12 enzymes were polymorphic; and the number of allozymes (mobility variants), which were equated with alleles, averaged 9.3 per locus in E. coli. For Shigella species, the mean number of alleles was 2.9 per locus. Some 77% of the allozymes recorded in Shigella were shared with E. coli. A total of 302 unique genotypic combinations of alleles over the 12 loci (electrophoretic types, ETs) was distinguished, of which 279 represented E. coli and 23 were Shigella. Among electrophoretic types, mean allelic diversity per locus was 0.52 for E. coli and 0.29 for Shigella. It was estimated that there are, on the average, about 0.3 detectable codon differences per locus between pairs of strains of E. coli and Shigella, which is roughly equivalent to 1.2 amino acid differences per enzyme. Evidence that the enzyme loci studied are a random sample of the genome is provided by a significant positive correlation between estimates of genetic divergence between pairs of strains obtained by DNA reassociation tests and estimates of genetic distance between the same strains based on electrophoresis. A principal components analysis of allozyme profiles revealed that the 302 ETs fall into three overlapping clusters, reflecting strong non-random associations of alleles, largely at four loci. Each of the four ETs of E. coli that have been most frequently recovered from natural populations has an allozyme profile that is very similar to, or identical with, the hypothetical modal ET of one of the groups. ETs of Shigella fall into two of the groups. No biological significance can at present bbe attributed to the genetic structure revealed by Multilocus electrophoretic techniques. The electrophoretic data are fully compatible with other molecular and more conventional evidence of a close affinity between E. coli and Shigella, and they raise questions regarding the present assignments of certain strains to species. In support of evidence from DNA reassociation tests and serotyping, the present study suggests that S. sonnei is homogeneous in chromosomal genotype.     

Expression of synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) in Escherichia coli

Synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) were assembled and cloned using established Escherichia coli expression plasmids. Transformed E. coli cells were able to synthesize either a fusion protein, comprising the first seven amino acids of beta-galactosidase, a linker fragment and bovine FGF, or genomic human bFGF. The two growth factors were purified from E. coli lysates by cation exchange and heparin-Sepharose affinity chromatography. The purified recombinant proteins were biologically active as monitored by their mitogenic activity for bovine aortic endothelial cells and their angiogenic capacity in the rabbit cornea.     

Comparison of the structures of L-glutamate decarboxylases from human and rat brains

Human and rat L-glutamate decarboxylases have been purified to electrophoretic homogeneity. These two enzymes were compared using an immunochemical method, amino acid analysis and tryptic fingerprinting. Structural studies revealed several differences in the primary structure of the two enzymes, but the immunochemical method used did not distinguish between the antigenicity of the two proteins.     

Pseudomonas putida Tryptophan Synthetase

The two protein components of Pseudomonas putida tryptophan synthetase have been purified to homogeneity. Although there is general similarity between the Pseudomonas enzyme and that of the enteric bacteria, many differences were found. Components from Escherichia coli and P. putida do not stimulate each other enzymatically, and the enzymes differ in their response to monovalent cations. Serine deamination occurs best with the intact enzyme of P. putida, not with the beta(2) subunit alone as in E. coli. The amino acid compositions of the alpha subunits differ appreciably. These findings extend earlier studies showing differences between enteric organisms and pseudomonads in the regulation and genetic organization of the enzymes of the tryptophan pathway.     

Isolation and characterization of bacterial flagellar hook proteins from salmonellae and Escherichia coli.

Flagellar hook proteins from Salmonella and Escherichia coli were dissociated in acid and purified by diethylamino-ethyl-cellulose column chromatography. These two proteins had the same electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. However, analytical electrofocusing patterns showed that these proteins had different isoelectric points (4.7 for Salmonella typhimurium and 4.4 for E. coli). Immunodiffusion and immuno-electron microscopy carried out with antisera prepared against purified hook proteins from S. typhimurium and E. coli showed that these antisera reacted with both hooks. Affinity chromatography allowed separation of antibodies specific for hook proteins from each bacterial species. These results indicate that the hook proteins share common antigenic determinants as well as specific antigens, although the specificity is not quantitatively resolved. From comparisons of the amino acid composition of the hook proteins and flagellins, it was concluded that the differences between flagellins from S. typhimurium and E. coli were larger than those between hook proteins from these species.     

Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli

Heterodimeric beta-galactosidase of Lactobacillus reuteri L103 is encoded by two overlapping genes, lacL and lacM. The lacL (1887bp) and lacM (960bp) genes encode polypeptides with calculated molecular masses of 73,620 and 35,682Da, respectively. The deduced amino acid sequences of lacL and lacM show significant identity with the sequences of beta-galactosidases from other lactobacilli and Escherichia coli. The coding regions of the lacLM genes were cloned and successfully overexpressed in E. coli using an expression system based on the T7 RNA polymerase promoter. Expression of lacL alone and coexpression of lacL and lacM as well as activity staining of both native and recombinant beta-galactosidases suggested a translational coupling between lacL and lacM, indicating that the formation of a functional beta-galactosidase requires both genes. Recombinant beta-galactosidase was purified to apparent homogeneity, characterized and compared with the native beta-galactosidase from L. reuteri L103.     

Cold-adapted beta-galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis

The beta-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH(2)-terminal amino acid sequence of the purified enzyme indicate that the beta-galactosidase subunit is composed of 1,038 amino acids with a calculated M(r) of 118,068. This beta-galactosidase shares structural properties with Escherichia coli beta-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis beta-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant beta-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis beta-galactosidase can outperform the current commercial beta-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted beta-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.     

Evolutionary relationships among bacterial carbamoyltransferases

An immunological approach was used for the study of ornithine carbamoyltransferase (OTCase) evolution in bacteria. Antisera were prepared against the anabolic and catabolic OTCases of Pseudomonas aeruginosa and Aeromonas formicans as well as against OTCase and putrescine carbamoyltransferases from Streptococcus faecalis; these antisera were then tested against the unpurified OTCases, either anabolic or catabolic, of 34 bacterial strains. Extensive cross-reactions were observed between the antisera to catabolic OTCases from P. aeruginosa, A. formicans and S. faecalis and the catabolic enzymes from other species or genera. These antisera cross-reacted also with the anabolic OTCases of strains of the Enterobacteriaceae but not with the anabolic OTCases of the same species or of other species or genera. The cross-reaction measured between the antisera against P. aeruginosa anabolic OTCase and the anabolic OTCases of other Pseudomonas were largely in agreement with the phylogenic subdivision of Pseudomonas proposed by N. J. Palleroni. The correlation was also significantly higher with the anabolic enzyme of an archaeobacterium, Methanobacterium thermoaceticum, than with the catabolic or anabolic OTCases from other genera in the eubacterial line. The antiserum raised against A. formicans anabolic OTCase was quite specific for its antigen and appeared to be raised against the heaviest of the various oligomeric structures of the enzyme.     

Expression of the lactose transposon Tn951 in Escherichia coli, Proteus and Pseudomonas

The control of beta-galactosidase specified by the lactose transposon Tn951 (inserted into RP1 to give pGC9114) has been studied in Escherichia coli K12, Proteus mirabilis, Pseudomonas aeruginosa and Pseudomonas putida; in the first two species comparison could be made with Flac. In E. coli K12, the Tn951 and chromosomally encoded enzymes showed marked qualitative differences in regulatio, the former giving a substantially lower maximum induced level and induction ratio. Several parameters were slightly affected by strain background. In P. mirabilis, beta-galactosidase control determined by both Flac (in accord with earlier work) and pGC9114 was markedly different from E. coli in that maximal induced levels were about an order of magnitude lower and the induction ratio was reduced to 3 to 5. In Ps. aeruginosa and Ps. putida, Tn951-specified lac expression was qualitatively similar to that in P. mirabilis. Possible reasons for anomalous expression in Proteus and Pseudomonas are discussed.     

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.     

A model for indication of DNA functional and structural changes at low-dose radiation

The work offers an "in vivo-in vitro" model which allows to identify DNA's both structural and functional damages caused by gamma-irradiation in doses from 0.02 to 0.25 Gy. As a donor the authors used an irradiated pTTQ 19 plasmide which had two marker genes: the ampicilline-resistant gene (amp(r)) and beta-galactosidase structural gene (lacZ alpha). E. coli GM 109 bacterial stamm transformed by the irradiated plasmide was used as a recipient. The structural damages of the irradiated plasmide were registered by DNA electrophoretic analysis in agarose gel. The plasmide DNA dysfunctions were assessed by its ability to pass on ampicilline resistance to E. coli bacterial cells as well as by beta-galactosidase level. The irradiated plasmide was found to have a tendency to decrease beta-galactosidase activity and number of E. coli ampicilline-resistant transformants depending on the received radiation dose: by 24.5% (0.05 Gy), 30.9% (0.19 Gy), and by 40.2% (0.25 Gy).     

Nucleotide sequence and analysis of the speA gene encoding biosynthetic arginine decarboxylase in Escherichia coli.

The DNA sequence of a 3.23-kilobase fragment of the Escherichia coli chromosome encoding biosynthetic arginine decarboxylase (ADC) was determined. This sequence contained the speA open reading frame (ORF) as well as partial speB and metK ORFs. The ADC ORF is 1,974 nucleotides long; the deduced polypeptide contains 658 amino acids with a molecular size of 73,980 daltons. The molecular weight and predicted ADC amino acid composition are nearly identical to the amino acid analysis of purified ADC performed by Wu and Morris (J. Biol. Chem. 248:1687-1695, 1973). A translational speA-lacZ fusion, pRM65, including 1,389 base pairs (463 amino acids) of the 5' end of speA was constructed. Western blots (immunoblots) with beta-galactosidase antisera revealed two ADC::beta-galactosidase fusion proteins in E. coli bearing pRM65: 160,000 and 156,000 daltons representing precursor and mature hybrid proteins, respectively. The predicted amino acid sequence of ADC contains a region of six amino acid residues found in two bacterial diaminopimelic acid decarboxylases and three eucaryotic ornithine decarboxylases. This conserved sequence is located approximately eight amino acids from the putative pyridoxal phosphate-binding site of ADC and is predicted to be involved in substrate binding.