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Stationary-phase acid resistance and injury of recent bovine Escherichia coli O157 and non-O157 biotype I Escherichia coli isolatest.

Stationary-phase acid resistance and the induction of acid resistance were assessed for recent bovine carcass isolates of Escherichia coli, including 39 serotype O157 strains and 20 non-O157 strains. When grown to stationary phase in the absence of glucose and without prior acid exposure, there was a range of responses to a pH challenge of 6 h at pH 2.5. However, populations of 53 of the 59 E. coli isolates examined were reduced by less than 2.00 log CFU/ml, and populations of 24 of these isolates were reduced by less than 1.00 log CFU/ml. In contrast, there was little variation in population reductions when the E. coli were grown with glucose and preadapted to acidic conditions. With few exceptions, acid adaptation improved survival to the acid challenge, with 57 of the 59 isolates exhibiting a log reduction of less than 0.50. Differences in acid resistance or the ability to adapt to acidic conditions between E. coli O157:H7 and non-O157 commensal E. coli were not observed. However, we did find that the E. coli O157 were disposed to greater acid injury after the low pH challenge than the non-O157 E. coli, both for cells that were and were not adapted to acidic conditions before the challenge. The enhancement of low pH survival after acid adaptation that was seen among these recent natural isolates of E. coli O157 further supports the idea that the previous environment of this pathogen should be a consideration when designing microbial safety strategies for foods preserved by low pH and acid.

Acetic Acid↗

Long-haul transport and lack of preconditioning increases fecal shedding of Escherichia coli and Escherichia coli O157:H7 by calves.

The effects of weaning and transport on fecal shedding of Escherichia coli and on E. coli O157:H7 were investigated using 80 Angus and 94 Charolais range steer calves blocked by breed and assigned to four treatments. The calves were or were not preconditioned before transport on commercial cattle liner to the feedlot via long (15 h) or short (3 h) hauling duration, yielding preconditioned long haul (P-L; n = 44), preconditioned short haul (P-S; n = 44), nonpreconditioned long haul (NP-L; n = 43), and nonpreconditioned short haul (NP-S; n = 43). Preconditioned calves were vaccinated and weaned 29 and 13 days, respectively, before transport. Nonpreconditioned calves were weaned 1 day before long or short hauling, penned for 24 h and hauled again for 2 h, and vaccinated on arrival at the feedlot. Fecal samples were collected from calves while on pasture, at weaning, at loading for transport, on arrival at the feedlot, twice in the first week, and on days 7, 14, 21, and 28 for enumeration of total E. coli (biotype 1) and detection of E. coli O157:H7. No calves were positive for E. coli O157:H7 before transport. Following transport, more (P < 0.005) NP-L calves (6 of 43) tested positive for E. coli O157:H7 than did P-L (1 of 44), NP-S (1 of 43), or P-S (0 of 44) calves, and on days 0, 1, 7, and 21, their levels of shedding of E. coli were higher (P < 0.005). The calves' susceptibility to infection from the environment (possibly the holding facilities or feedlot pens) was likely elevated by the stresses of weaning, transport, and relocation. Lack of preconditioning and long periods of transport (NP-L) increased fecal shedding of E. coli and E. coli O157:H7. Preconditioning may serve to reduce E. coli O157:H7 shedding by range calves on arrival at the feedlot.

Animals↗

Preharvest evaluation of coliforms, Escherichia coli, Salmonella, and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers.

Microbiological analyses of fresh fruits and vegetables produced by organic and conventional farmers in Minnesota were conducted to determine the coliform count and the prevalence of Escherichia coli, Salmonella, and E. coli O157:H7. A total of 476 and 129 produce samples were collected from 32 organic and 8 conventional farms, respectively. The samples included tomatoes, leafy greens, lettuce, green peppers, cabbage, cucumbers, broccoli, strawberries, apples, and seven other types of produce. The numbers of fruits and vegetables was influenced by their availability at participating farms and varied from 11 strawberry samples to 108 tomato samples. Among the organic farms, eight were certified by accredited agencies and the rest reported the use of organic practices. All organic farms used aged or composted animal manure as fertilizer. The average coliform counts in both organic and conventional produce were 2.9 log most probable number per g. The percentages of E. coli-positive samples in conventional and organic produce were 1.6 and 9.7%, respectively. However, the E. coli prevalence in certified organic produce was 4.3%, a level not statistically different from that in conventional samples. Organic lettuce had the largest prevalence of E. coli (22.4%) compared with other produce types. Organic samples from farms that used manure or compost aged less than 12 months had a prevalence of E. coli 19 times greater than that of farms that used older materials. Serotype O157:H7 was not detected in any produce samples, but Salmonella was isolated from one organic lettuce and one organic green pepper. These results provide the first microbiological assessment of organic fruits and vegetables at the farm level.

Escherichia coli↗

Studies to select appropriate nonpathogenic surrogate Escherichia coli strains for potential use in place of Escherichia coli O157:H7 and salmonella in pilot plant studiest.

The response of a potential nonpathogenic surrogate organism to a particular treatment should closely mimic the response of the target pathogenic organism. In this study, growth characteristics (generation time, lag phase duration, and maximum population), pH at stationary phase, and survival characteristics (level of attachment and survival on apple surfaces, resistance to hydrogen peroxide decontamination treatments, and thermal resistance at 60 degrees C) of 15 nonpathogenic generic Escherichia coli strains and one nonpathogenic E. coli O157:H43 strain were compared with those of two E. coli O157:H7 strains and two Salmonella strains. Few differences in growth characteristics or pH at stationary phase were evident between nonpathogenic and pathogenic strains tested. However, considerably more separation among strains was seen following investigation of survival characteristics. E. coli ECRC 97.0152, which does not contain genes encoding for known virulence factors associated with E. coli O157:H7, appears to be a good surrogate candidate, with growth and survival characteristics similar to those of E. coli O157:H7 strains. The less heat-resistant surrogate strains E. coli NRRL B-766 and NRRL B-3054 and E. coli ATCC 11775, ATCC 25253, and ATCC 25922 may be used when attempting to model the heat resistance of Salmonella Montevideo G4639 and Salmonella Poona RM 2350, respectively. These surrogate strains may be useful for evaluating the efficacy of intervention steps in reducing populations of selected strains of E. coli O157:H7 and Salmonella in processing environments where these pathogens cannot be introduced.

Adaptation, Physiological↗

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland.

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.

Abattoirs↗

Incidence of enterohemorrhagic Escherichia coli, Escherichia coli O157, Salmonella, and Listeria monocytogenes in retail fresh ground beef, sprouts, and mushrooms.

The objective of this study was to determine the prevalence of enterohemorrhagic Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef (1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were collected over a 12-month period and analyzed for the presence of these pathogens. PCR assays, followed by culture confirmation were used to determine the presence or absence of each organism. Of the 1,750 ground beef samples analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L. monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3 (1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%) were positive for Salmonella and none were positive for L. monocytogenes. Among the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4 samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.

Agaricales↗

[Verotoxin-producing Escherichia coli (VTEC) and Escherichia coli O157:H7 in medicine and food industry].

Escherichia coli O157:H7 is now recognised as an important human pathogen. Illness caused by E. coli O157:H7 infection can range from self limited, watery diarrhea to life-threatening manifestations such as hemorrhagic colitis, hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. The mode of transmission is primarily through food (e.g. undercooked minced beef products, especially beef burgers, raw cows' milk and cheese, contaminated pasteurised milk and untreated water.). Studies to date indicate that cattle is an important reservoir of the organism. Public health measures to control VTEC infection are broadly similar to the measures needed to control other gastro-intestinal infections. Because of the potential low infection dose, laboratory diagnosis of O157 VTEC in food samples has developed over recent years with the use of liquid enrichment and the development of methods such as immunomagnetic separation. VTEC of other serogroups than O157 have no reliable biochemical, serological or morphological characteristics (other than VT production itself) to distinguish them from commensal E. coli. Thus to detect VTEC other than O157 and phenotypic variants of E. coli O157 in food, we have to use methods for detection of verocytotoxin production and VT genes.

Animals↗

Virulence factors of Escherichia coli. IV. Association in Escherichia coli of LD50 with haemolysin production, haemagglutinating capacity, antigens K1, K5 colicinogenicity and pathogenicity.

Haemolysin production (HLY), mannose resistant haemagglutinating activity (MRA), presence of antigens K1 and K5 and colicinogenicity (Col) were compared with LD50 for mice in 663 Escherichia coli strains, including 281 faecal, 129 urinary and 253 other extraintestinal isolates. Those isolates that LD50 value fell into less than or equal to 10(6) LD50 category were arbitrarily termed highly virulent (HV) and those which belonged to greater than or equal to 10(7) LD50 category were considered avirulent (AV). HV isolates occurred significantly more frequently (58%) among strains from different extraintestinal samples than from faeces (14%) or urine (16%). The incidence of HV strains was significantly higher in patients with sepsis (43%) or meningitis (100%) than in patients with enteritis (20%), urinary tract infections (UTI, 16%) or in healthy subjects (28%). The incidence of HV strains in the most frequent (O1, O2, O4, O6, O7, O18, O75) serogroups was significantly higher (60%) than in others (10%). Strains with different virulence markers (HLY, MRA, K1, K5, Col) belonged significantly more frequently to the HV group than those which failed to have these markers (44 vs 27%, 51 vs 25%, 83 vs 17%, 78 vs 27%, 52 vs 16%, respectively). Important role of antigen K1 playing in pathomechanism of meningitis was confirmed by data of analysis according to which significant difference was revealed in the incidence of HV strains between groups of isolates with MRA+K1+ (71%) and MRA+K1- (44%, p less than 0.02), or between groups of isolates with MRA+K1- and MRA-K1+ (91%, p less than 0.001). Moreover there were significant differences in the incidences of HV strains in K1+Col- (73%) and K1-Col+ (29%, p less than 0.001), and in K1+Col+ (86%) and K1-Col+ (29%, p less than 0.001) groups. Further evidence was given by those data that there were no significant differences between groups of HV strains with MRA+K1+ and MRA-K1+ (p greater than 0.05) or with K1+Col+ and K1+Col- (p greater than 0.1) properties. Isolates that possessed simultaneously two of MRA, HLY, Col markers were more pathogenic in LD50 assay than those that had one or the other of these markers alone. Strains in serogroup O18 killed mice significantly more frequently than those of other serogroups independently of having any virulence factor, suggesting that bacteria in serogroups O18 must have some special virulence other than K1, Col, MRA or K5. MRA+HLY+ HV strains occurred frequently in extraintestinal diseases (42%) supporting the preconception that these properties play an important role also in the pathomechanism of extraintestinal infections other than UTI.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Bacterial↗

Pathogenic effect of enterotoxigenic Escherichia coli and Escherichia coli causing infantile diarrhoea.

Macroscopic, light and electron microscopic alterations in ligated rabbit intestinal loops challenged with five standard enterotoxigenic Escherichia coli (ETEC) and twenty-three enteropathogenic E. coli (EEC-I) strains, freshly isolated from infantile enteritis cases, were investigated. Only two O26 : K60 : H11 strains produced enterotoxin. Their living cultures, sterile filtrates of the fluid medium and ultrasonic lysates of the bacteria resulted in pronounced hypersecretion of the intestinal epithelium followed by fluid accumulation and loop dilatation. These two E. coli strains, similarly as the other loop-negative EEC-I strains, were able to penetrate into the intestinal epithelium. In contrast to the standard ETEC strains, the EEC-I bacteria, adhering to the brush border, intruded into the microvilli, multiplied on the outer epithelial cell membrane making close contact with it and, causing, shedding of microvilli, penetrated into enterocytes becoming enclosed in membrane-bound phagosome-like vacuoles, appeared in the lamina propria and elicited mild focal polymorphonuclear infiltration.

Animals↗

Virulence factors of Escherichia coli. III. Correlation with Escherichia coli pathogenicity of haemolysin production, haemagglutinating capacity, antigens K1, K5, and colicinogenicity.

A total of 1156 Escherichia coli strains including 489 faecal, 384 urinary, 283 other extraintestinal isolates was compared for haemolysin production (Hly), mannose resistant haemagglutinating activity (MRA), presence of antigens K1 and K5 and colicinogenicity (Col). K1 capsule which was demonstrated only in a few serogroups (O1, O2, O7, O18) occurred more frequently among extraintestinal (32.1%) than among faecal (4.3%) or urinary (7.3%) isolates. In the incidence of antigen K5 there was no difference between faecal and urinary (3.3%; 3.1%) or between urinary and other extraintestinal (5.3%) isolates belonging mainly to serogroups O2, O6, O18 and O75. Col+ isolates occurred frequently in all samples (23.5% of faecal, 31.7% of urinary and 43.4% of other extraintestinal strains), they being significantly more frequent in serogroups O1, O2, O7, O18 than in others. A close association existed between K1+ and Col+ properties, mainly (24.4%) among strains isolated from extraintestinal sources other than urine. The frequent coexistence of K1+ and Col+ in serogroups O1, O2, O7, O18 offers a further explanation for the extraintestinal pathogenicity of these serogroups. Neither Hly+ and K1+, nor Hly+ and Col+ were associated. MRA+ and K1+ correlated mainly in serogroups O1, O2 but never occurred simultaneously in serogroup O18. Connection between MRA+ and Hly+ was not associated with other virulence factors (K1, Col). The results showing a close connection among certain serogroups (O1, O2, O4, O6, O7, O18) and certain markers of pathogenicity (MRA, Hly, K1, Col) support the concept that E. coli strains have a clonal connection.

Antigens, Bacterial↗

Administration of purified colonization factor antigens (CFA/I, CFA/II) of enterotoxigenic Escherichia coli to volunteers. Response to challenge with virulent enterotoxigenic Escherichia coli.

Colonization factor antigens (CFAs) were administered orally to volunteers, and the mucosal immune response was assessed by measuring secretory immunoglobulin A (IgA) in saliva and intestinal secretions and by challenge with virulent enterotoxigenic Escherichia coli (ETEC). A combination of CFA/I and CFA/II (8 mg each) administered orally in four doses in milk failed to induce a mucosal IgA response and also failed to protect against challenge with CFA-positive ETEC. When CFA was administered orally (1.5 mg divided into three doses with bicarbonate) to volunteers without preexisting serum anti-CFA levels, it failed to elicit a serum IgG or intestinal secretory IgA response or to protect against challenge with virulent ETEC. The CFA is destroyed by acid gastric contents but it induced significant antibody titer rises in 8 of 11 (73%) volunteers with preexisting serum anti-CFA IgG levels after oral administration of 1 mg of CFA/I with sodium bicarbonate. Subcutaneous priming (50 micrograms CFA/I) did not induce a serum IgG response but, when followed by oral boosting (1 mg CFA/I in two divided doses with sodium bicarbonate), induced intestinal anti-CFA secretory IgA in 4 of 8 volunteers and protected against challenge with CFA/I-positive ETEC. These results, although preliminary, are encouraging and demonstrate that it may be possible to develop an effective oral vaccine based on soluble nonreplicating antigens such as purified CFAs.

Administration, Oral↗

Interim guidelines for the control of infections with Vero cytotoxin producing Escherichia coli (VTEC). Subcommittee of the PHLS Working Group on Vero cytotoxin producing Escherichia coli (VTEC).

The threat to public health in the United Kingdom of infections with Vero cytotoxin producing Escherichia coli (VTEC), and E. coli O157 in particular, has led the PHLS to develop guidance for control. Published and unpublished data have been reviewed by a subcommittee of the PHLS working group on VTEC. This paper summarises the review and presents interim guidelines for the control of VTEC infections.

Adult↗

Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5 alpha.

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Animals↗

Prevention and control of enterohaemorrhagic Escherichia coli (EHEC) infections: memorandum from a WHO meeting. WHO Consultation on Prevention and Control of Enterohaemorrhagic Escherichia coli (EHEC) Infections.

Escherichia coli is a commonly occurring inhabitant of the intestine of humans and other animals, but there are several pathogenic types of E. coli which cause a variety of human diseases. One of these pathogenic types, E. coli O157:H7, belongs to the group of enterohaemorrhagic E. coli (EHEC) which produce potent toxins and cause a particularly severe form of disease, haemorrhagic colitis (HC). About 10% of patients with HC can go on to develop haemolytic uraemic syndrome (HUS), a life-threatening complication of E. coli O157:H7 infection that is characterized by acute renal failure, haemolytic anaemia, and thrombocytopenia. These sequelae are particularly serious in young children and older people. On average, 2-7% of patients with HUS die, but in some outbreaks among the elderly the mortality rate has been as high as 50%. This Memorandum reviews the growing importance of E. coli O157:H7 as a foodborne pathogen and reports on the issues of surveillance, outbreak investigation, and control strategies with respect to EHEC infections that were discussed at the WHO Consultation on Prevention and Control of EHEC Infections, held in Geneva on 28 April to 1 May 1997. Recommended measures for prevention and control include the following: use of potable water in food production; presentation of clean animals at slaughter; improved hygiene throughout the slaughter process; appropriate use of food processing measures; thorough cooking of foods; and the education of food handlers, abattoir workers, and farm workers on the principles and application of food hygiene.

Child↗

Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2DPAGE database.

Numerous Escherichia coli proteins have already been characterized by two-dimensional gel electrophoresis (2-D PAGE), using carrier ampholytes in the first dimension (VanBogelen, R. A., Sankar, P., Clark, R. L., Bogan, J. A. and Neidhardt, F. C., Electrophoresis 1992, 13, 1014-1054). We present here a reference protein map of E. coli obtained with immobilized pH gradients (IPG) and available in a SWISS-2DPAGE format. Out of the protein spots identified in the E. coli gene protein database by Neidhardt's group, 153 have been identified in the E. coli gene protein database by Neihardt's group, 153 have been identified on the E. coli SWISS-2DPAGE database map by gel comparison and most of them were confirmed either by the analysis of amino acid composition (AAC) and/or N-terminal microsequencing. Additionally, five as yet unsequenced proteins were found. The E. coli SWISS-2DPAGE database is part of the ExPASy molecular biology server accessible through the Word Wide Web network.

Amino Acid Sequence↗

Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K-12.

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli K1 strain to an E coli K-12 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to "authentic" protein K when chymotryptic peptides of 125I-labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ-473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.

Bacterial Outer Membrane Proteins↗

Studies of colicin action on wall-less stable L-forms of Escherichia coli. II. Growth inhibition of complete and wall-less (L-form) cells of Escherichia coli by basic colicin types.

The inhibitive activity of colicins of 16 types (produced by 22 colicinogenic strains) on rods and protoplast-like stable L-form cells of the strains Escherichia coli B, W1655 F+ and W1655 F- was compared. The results of 58 combinations tested fall into four groups (with three subgroups): both cell forms are sensitive: a) both cell forms are about equally sensitive, b) rods are distinctly more sensitive than L-form cells, c) L-form cells are distinctly more sensitive than rods; only rods are sensitive, L-form cells are not sensitive; only L-form cells are sensitive, rods are not sensitive; neither cell form is sensitive. Group la represents simple sensitivity; both cell wall and cytoplasmic membrane receptors are present and functioning. Groups 1b and 2 represent sensitivity, substantially or completely mediated by cell wall receptors. Groups 1c and 3 represent partial or complete "pseudoresistance" or "pseudotolerance"; cell wall receptors are absent or non-lethal, but cytoplasmic membrane ones are present and mediate the lethal effect. Group 4 represents both true resistance and true tolerance.

Colicins↗

Redesign of the substrate specificity of Escherichia coli aspartate aminotransferase to that of Escherichia coli tyrosine aminotransferase by homology modeling and site-directed mutagenesis.

Although several high-resolution X-ray crystallographic structures have been determined for Escherichia coli aspartate aminotransferase (eAATase), efforts to crystallize E. coli tyrosine aminotransferase (eTATase) have been unsuccessful. Sequence alignment analyses of eTATase and eAATase show 43% sequence identity and 72% sequence similarity, allowing for conservative substitutions. The high similarity of the two sequences indicates that both enzymes must have similar secondary and tertiary structures. Six active site residues of eAATase were targeted by homology modeling as being important for aromatic amino acid reactivity with eTATase. Two of these positions (Thr 109 and Asn 297) are invariant in all known aspartate aminotransferase enzymes, but differ in eTATase (Ser 109 and Ser 297). The other four positions (Val 39, Lys 41, Thr 47, and Asn 69) line the active site pocket of eAATase and are replaced by amino acids with more hydrophobic side chains in eTATase (Leu 39, Tyr 41, Ile 47, and Leu 69). These six positions in eAATase were mutated by site-directed mutagenesis to the corresponding amino acids found in eTATase in an attempt to redesign the substrate specificity of eAATase to that of eTATase. Five combinations of the individual mutations were obtained from mutagenesis reactions. The redesigned eAATase mutant containing all six mutations (Hex) displays second-order rate constants for the transamination of aspartate and phenylalanine that are within an order of magnitude of those observed for eTATase. Thus, the reactivity of eAATase with phenylalanine was increased by over three orders of magnitude without sacrificing the high transamination activity with aspartate observed for both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Aminotransferases↗