Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature.

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.

Induced hypocalcaemia by Na2EDTA infusion. A review.

Disodium ethylenediaminetetraacetate (Na2EDTA) has been used in infusion studies in cows and other species for more than 35 years, primarily for the induction of hypocalcemia as a model for milk fever. This paper reviews such studies and discusses blood calcium kinetics, toxicology, changes in various blood parameters and the effect on blood circulation, cardiac function and smooth muscle motility in the gastro-intestinal tract and in the pregnant uterus. It is concluded that Na2EDTA infusion may serve as a valid model for spontaneous hypocalcemia. However, experimental results may vary with factors such as the choice of method for blood total calcium analysis, and the rate of EDTA infusion. Standardization of these and certain other experimental conditions may greatly improve the comparability of results obtained in EDTA infusion studies. For cows, an infusion rate of 1.2 ml/kg/h of a 5% (w/v) solution, corresponding to 0.25 mmol/kg/min, has been suggested as a standard infusion.

Rumen motility during induced hyper- and hypocalcaemia.

Rumen motility was recorded on an experimental cow by means of telemetric signal transfer from strain gauge force transducers fixed surgically on the peritoneal surface of the rumen wall in the left flank. The normocalcaemic cow was given a standard milk fever treatment with calcium borogluconate (400 ml with 14 mg Ca/ml) intravenously. Transient clinical signs were: decreased rumination, muscle ticks, salivation and a heart rate reduction of 20%. Rectal temperature remained unaltered. Frequency of rumen contractions was reduced up to 40% whereas amplitude of contractions did not deviate from baseline values. Hypocalcaemia was induced in a second experiment by iv infusion of Na2EDTA. At 0.60 mmol/l ionized blood calcium periods of no motility were recorded whereas inactivity of rumen activity was persistent at 0.55 mmol/l ionized blood calcium. The cow went down at 0.45-0.48 mmol/l ionized blood calcium at which point the heart rate was increased by 40%. The high sensitivity of the method employed allowed the conclusion that already at a concentration of ionized blood calcium at 1.0 mmol/l both frequency and amplitude of rumen contractions decreased rapidly although eating behaviour and rumination appeared unaffected during the short term observation periods. Implications of this finding towards health and production in transition cows are discussed.

Determination of the recognition sequence of the type II restriction endonuclease, LlaCI, from Lactococcus lactis W15.

A new type II restriction endonuclease, called LlaCI, was partially purified from Lactococcus lactis subsp. cremoris W15. The characterisation of the LlaCI endonuclease showed it to be an isoschizomer of HindIII, recognising the sequence 5-'A decreases AGCTT-3'. The cleavage site is indicated by the arrow.

Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp. cremoris W56.

The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp. cremoris W56 and sequenced. The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa). A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L. lactis.

Tandem translation of Bacillus subtilis initiation factor IF2 in E. coli. Over-expression of infBB.su in E. coli and purification of alpha- and beta-forms of IF2B.su.

The protein synthesis initiation factor, IF2, in Bacillus subtilis has previously been characterized as being present in two forms, alpha and beta, of molecular mass 79 and 68 kDa, respectively, on the basis of their cross-reaction with anti-E. coli IF2 antibodies and by the DNA sequence of the gene for IF2, infBB.su. In this work we have cloned infBB.su in E. coli cells. Two proteins of molecular mass identical to the B. subtilis IF2 alpha and -beta were over-expressed and purified using a new three-step ion-exchange chromatography procedure. The N-terminal amino acid sequence of the two proteins was determined and the results confirmed that the two forms were IF2 alpha and -beta, both encoded by the infB gene. The N-terminal amino acid sequence determined for IF2 beta is Met94-Gln-Asn-Asn-Gln-Phe. The presence of methionine at position 94 shows that this form is, in fact, the result of a second translational initiation in infBB.su mRNA, since the codon at amino acid position 94 is GUG, which is the normal codon for valine, but also known to be an initiator codon. This is a new example of the unusual tandem translation in E. coli of an open mRNA reading frame.

Tandem translation of E. coli initiation factor IF2 beta: purification and characterization in vitro of two active forms.

Two forms of E. coli initiation factor IF2, IF2 alpha and IF2 beta, have been known for several years. Both forms are products of the gene infB with translational initiation at codon 1 (AUG) and codon 158 (GUG) in the same reading frame. In this work we demonstrate that IF2 beta exists in two forms, IF2 beta and IF2 beta' with initiation codons 158 (GUG) and 165 (AUG) and molecular masses of 79.7 kDa and 78.8 kDa respectively. We have recently described a fast purification method for IF2 alpha, using an FPLC procedure consisting of ion-exchange liquid chromatography on Q Sepharose HP, Mono Q and Mono S. After the Mono Q step, an apparently homogeneous IF2 beta was observed when analyzed by SDS-PAGE. However the chromatography on Mono S results in the elution of two peaks containing IF2 beta. The N-terminal amino acid sequence of the two proteins identified the first peak to be IF2 beta and the second as a protein which we term IF2 beta' starting seven residues downstream at the AUG codon 165. The activity in vitro of the two purified forms of IF2 beta was tested by measuring the stimulation of binding of the initiator fMet-tRNA(fMet) to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger-RNA. In this assay no difference in activity is detected.

Superexpression and fast purification of E coli initiation factor IF2.

For the production of large quantities of E coli initiation factor IF2 we have constructed an improved overexpression system. The gene infB was cloned into the thermo-inducible runaway plasmid pCP40 [1] and subsequently transformed into the E coli strain C600[pcI857]. In this system the expression of infB is under the control of the strong promoter lambda PL and the cells carry the plasmid pcI857, which contains a thermosensible lambda cI repressor. Overexpression of IF2, which is approximately 30 times higher than the expression in wild-type-cells, is induced at 42 degrees C and continues for 2 h at 37 degrees C. From these cells pure and active IF2 was obtained using a novel 3-step FPLC-procedure consisting of ion-exchange liquid chromatography on Q-sepharose HP, MonoQ and MonoS. In approximately 8 h, 5 mg of pure and active IF2 can be obtained from 10 g overproducing cells. This corresponds to 5 mg of IF2 per litre of medium. The purification was monitored by Western immunoblotting and the activity of the purified factor was tested by measuring the stimulation of binding of the initiator fMet-tRNA(Met)f to 70S ribosomes in the presence of GTP and poly(A,U,G) as messenger RNA. Compared with previous methods our purification procedure avoids the use of materials such as DEAE-cellulose and phosphocellulose which have relatively poor flow rates. In addition to the higher flow capacity of Q-sepharose HP, this new matrix can be loaded with an S30 supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)