Beta lactamase

Biochem J, 1981 Jul 1, 197(1), 105 - 9
Denaturation of proteins and nucleic acids by thermal-gradient electrophoresis; Thatcher DR et al.; A polyacrylamide-gel-electrophoresis method has been developed that permits the analysis of conformational changes that occur during the thermal denaturation of macromolecules . A stable transverse temperature gradient was produced in an aluminium heating jacket clamped around a vertical polyacrylamide slab gel . After temperature equilibration, gels were loaded with either a layer of protein solution (20-200 micrograms/gel) or a solution of double-stranded DNA (20 micrograms/gel) and electrophoresis begun . At the end of the run the gels were stained and the effect of temperature on mobility observed . The technique proved informative both for the irreversible unfolding of proteins (Drosophila alcohol dehydrogenase and lactic acid dehydrogenase) and for a protein that was reversibly denatured by heat (beta-lactamase) . In the latter case a clear transition between the native enzyme and a slower-migrating denatured state was observed . The patterns obtained were analogous to the type produced by the transverse-urea-gradient-electrophoretic method of Creighton {(1979) J . Mol . Biol . 129, 253-264} . The method also resolved a complex mixture of double-stranded-DNA restriction-digest fragments.

Biochemistry, 1981 Jun 23, 20(13), 3688 - 95
Penicillanic acid sulfone: interaction with RTEM beta-lactamase from Escherichia coli at different pH values; Kemal C et al.; The interaction of the sulfone of penicillanic acid with the TEM-2 beta-lactamase from Escherichia coli has been investigated as a function of pH between pH 7.0 and 9.6 . The first-formed acyl-enzyme suffers one of three fates: deacylation, tautomerization to a bound enamine that transiently inhibited the enzyme, and a process (possibly transimination) that leads to enzyme inactivation . The observed changes in ultraviolet absorbance are consistent with the initially observed product of deacylation being the enamine tautomer (4) of the imine from malonsemialdehyde and penicillamine sulfinate . The same enamine can be generated nonenzymically from the sulfone at high pH . The transiently inhibited enzyme appears to be the same enamine attached to the enzyme by an ester linkage . The rather complex kinetic behavior can be deconvuluted by exploiting the effect of pH on the partitioning of the acyl-enzyme between deacylation and the transiently inhibited form of the enzyme . The pathways followed by penicillanic acid sulfone provide a model for the behavior of a number of other reagents that inactivate the beta-lactamase.

Biochemistry, 1981 Jun 23, 20(13), 3680 - 7
Penicillanic acid sulfone: an unexpected isotope effect in the interaction of 6 alpha- and 6 beta-monodeuterio and of 6,6-dideuterio derivatives with RTEM beta-lactamase from Escherichia coli; Brenner DG et al.; Penicillanic acid sulfone (1) is both a substrate and an inactivator of the RTEM beta-lactamase . About 7000 hydrolytic events occur before enzyme inactivation . The 6,6-dideuterio sulfone shows a 3-fold acceleration of both the hydrolysis reaction and the enzyme inactivation . The kinetic and spectroscopic results are nicely accommodated by a scheme in which a transiently stable intermediate is formed in an isotopically sensitive step . The deuterated material partitions less readily toward this transiently stable intermediate by virtue of a primary isotope effect, and more enzyme is then available for the hydrolysis and inactivation pathways . Use of the stereospecifically monodeuterated sulfones shows that the 6 beta hydrogen is preferentially abstracted in the formation of the transiently stable intermediate and allows a detailed picture of the interaction of the sulfone and the beta-lactamase to be drawn . The crystal structures of both the labeled and unlabeled compounds are reported.

Nucleic Acids Res, 1981 Jun 11, 9(11), 2517 - 33
Characterization of the beta-lactamase promoter of pBR322; Russell DR et al.; The beta-lactamase promoter of pBR322, derived from Tn3, has been characterized using several techniques . The transcription initiation site is located 35 base pairs from the translation initiation codon of beta-lactamase . The mRNA produced in vitro has a 5' pppGpA terminus . RNA polymerase bound at this start site protects a region from about -50 to +20 from DNase I cleavage using the footprinting technique . RNA polymerase binds rapidly to the beta-lactamase promoter . The half-time of association is less than one-half minute . The half-time of dissociation is approximately 6 hr . A study of the binding of RNA polymerase at different temperatures showed a large change between 11 degrees and 15 degrees C . Comparison of these parameters with those reported for other promoters is discussed.

Mutat Res, 1981 Jun, 82(1), 47 - 57
Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo{alpha}pyrene-7,8-dihydrodiol-9,10-oxide in Escherichia coli; Mizusawa H et al.; Plasmid-mediated transformation and mutagenesis induced by (+/-)-trans-benzo{alpha}pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E . coli) have been studied . Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells . Plasmid pK0482 DNA, which has two dominant genes, beta-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E . coli N99, AB1157, AB2463(recA-) and AB1886(uvrA-) . Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene . (1) Approx . 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA . (2) In Ab1886(uvrA-) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157 . (3) In AB2463(recA-), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed . (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.

Biochemistry, 1981 May 26, 20(11), 3214 - 9
Inactivation of RTEM beta-lactamase from Escherichia coli by clavulanic acid and 9-deoxyclavulanic acid; Charnas RL et al.; The interaction of the TEM-2 beta-lactamase with 9-deoxyclavulanic acid (3) and with both extensively labeled (2) and specifically labeled (1) clavulanic acid has been studied . The close similarity between 9-doexyclavulanate and clavulanate in kinetics, spectroscopic, and protein chemical terms show that the allyl alcohol group of clavulanate is irrelevant to its action as a beta-lactamase inactivator . Use of the radiolabeled samples of clavulanate shows that, of three irreversibly inactivated forms of the enzymes, two contain the whole clavulanate skeleton and the third only retains the carbon atoms of the original beta-lactam ring . These findings allow the complex interaction between clavulanic acid and the beta-lactamase to be defined more narrowly in chemical terms.

Biochemistry, 1981 May 12, 20(10), 2732 - 7
Inhibition of the RTEM beta-lactamase from Escherichia coli . Interaction of enzyme with derivatives of olivanic acid; Charnas RL et al.; The interaction of the RTEM beta-lactamase with two derivatives of olivanic acid has been studied . The compound MM22382 (1) behaves simply as a good substrate for the enzyme and is a relatively ineffective inhibitor . In contrast, the sulfate ester MM13902 (2) is a poor substrate and an excellent inhibitor of the enzyme . The inhibition derives from a branching of the normal hydrolytic pathway of the enzyme . At long times, all the catalytic activity of the enzyme returns . Free sulfate ion is not produced during the interaction with the enzyme, which rules out a mechanistic pathway involving beta elimination between C-6 and C-8 . The validity of a number of alternative schemes is assessed.

Biochemistry, 1981 May 12, 20(10), 2726 - 31
Inactivation of the RTEM beta-lactamase from Escherichia coli . Interaction of penam sulfones with enzyme; Fisher J et al.; The characteristics of the reaction of a number of mechanism-based inactivators of the RTEM beta-lactamase have suggested that a common mechanistic pathway may be followed by many of these compounds . These ideas have been tested by the synthesis and evaluation of some penam sulfones as beta-lactamase inactivators . The sulfones of poor beta-lactamase substrates are, as predicted, potent inactivators of the enzyme . A unique serin residue (Ser-70) is labeled by quinacillin sulfone, and it is likely that this serine acts nucleophilically in the normal hydrolytic reaction of the beta-lactamase to form an acyl-enzyme intermediate.

Antimicrob Agents Chemother, 1981 Apr, 19(4), 620 - 4
Latex agglutination inhibition card test for gentamicin assay: clinical evaluation and comparison with radioimmunoassay and bioassay; Standiford HC et al.; Gentamicin levels were determined in 100 serum specimens by a new latex agglutination inhibition card test, a radioimmunoassay (RIA), and a bioassay . Correlation coefficients determined by linear regression analysis demonstrated that the levels obtained by the latex agglutination inhibition card test had a high degree of correlation with the RIA and could be performed much faster and more economically when processing small numbers of specimens . The bioassay had a slightly lower degree of correlation with both the RIA and the latex test and was adversely influenced by concurrently administered antibiotics which could not be eliminated by beta-lactamase . When measuring gentamicin concentrations above 2 micrograms/ml, the coefficient of variation was less than 14% for the latex agglutination assay compared with 15% for the bioassay and 12% for RIA . The latex agglutination inhibition card test is a rapid, accurate, specific, and reproducible method for monitoring gentamicin levels in patients and is particularly applicable for laboratories processing small numbers of specimens.

Nature, 1981 Mar 19, 290(5803), 221 - 5
The E . coli beta-lactamase attenuator mediates growth rate-dependent regulation; Jaurin B et al.; We have identified a new control or attenuator region in the chromosomal beta-lactamase operon of Escherichia coli . A single base alteration within its attenuator led to a loss in the cell's ability to coordinate its content of beta-lactamase with growth rate . We suggest a mechanism through which this mode of regulation operates.

J Bacteriol, 1981 Mar, 145(3), 1310 - 6
Functional organization of plasmid pKM101; Langer PJ et al.; Tn5 insertion mutants and in vitro-generated deletion mutants of the mutagenesis-enhancing plasmid pKM101 have been used to identify several genetic regions on the pKM101 map . In clockwise order on the pKM101 map are: (i) the bla gene, coding for a beta-lactamase; (ii) the Slo region, responsible for retarding cell growth on minimal medium; (iii) the tra genes, enabling pKM101 to transfer conjugally; (iv) sensitivity to IKe phage (this function{s} maps within the tra region); (v) the muc gene(s), responsible for enhancing ultraviolet light and chemically induced mutagenesis in the cell; and (vi) the Rep region, essential for plasmid replication . The muc gene(s) and the Rep region are contained in a deoxyribonucleic acid region bounded by inverted repeated sequences.

Nucleic Acids Res, 1981 Jan 10, 9(1), 19 - 30
Cloning of bovine growth hormone gene and its expression in bacteria; Keshet E et al.; A hybrid plasmid was constructed containing beta-lactamase gene of plasmid pBR322 and cloned coding sequences of bovine growth hormone (BGH) . The constructed plasmid contains all DNA sequences required to encode BGH, and when used as a hybridization probe it detects one growth hormone gene in the bovine genome . The cloned DNA sequences are inserted into the beta-lactamase gene in the correct reading frame for BGH synthesis . The hybrid gene is expressed in bacteria and the product, a fused beta-lactamase-bovine growth hormone protein, is specifically immunoprecipitated with anti-serum to BGH . Unlike beta-lactamase, very little growth hormone containing sequences can be detected in the periplasmic space.

Acta Chem Scand B, 1981, 35(8), 567 - 72
Comparison of periplasmic and membrane associated beta-lactamase; Mantsala P et al.; Beta-lactamase encoded by a plasmid pBR 322 was produced during the active growth phases of Escherichia coli IA 199 . The maximal specific activity was about 15 times higher in shock fluid than in the cells disrupted by sonic disintegration . beta-Lactamase activity found in the membrane preparations increased gradually parallel to the cell growth . The amount of beta-lactamase in the membrane fraction, however, was only 0.2-0.4% of that found in shock fluid . beta-Lactamase was purified to homogeneity from shock fluid by a one-step procedure in a DEAE-Sepharose column . Most of the beta-lactamase activity present in the membrane fraction was released by salt extraction . beta-Lactamase solubilized treatment after salt extractions had the same molecular weight and immunological properties as beta-lactamase purified from the periplasmic space . Membrane associated beta-lactamase did not contain any covalently linked phospholipid.

Boll Ist Sieroter Milan, 1981, 60(1), 77 - 8
Legionellosis in an infant: first case in Italy; Fumarola D et al.; Among several children admitted in Hospital with febrile acute pneumonia, it has been found - by indirect immunofluorescence assay a significant seroconversion for Legionella pneumophila serogroup 1, compatible with a recent infection, in an 18-month-old boy . The clinical course of the disease was favourable, and a prompt recovery as observed after administration of an antibiotic (cefuroxime) stable to the bacterial beta-lactamase.

Proc Natl Acad Sci U S A, 1981 Jan, 78(1), 167 - 71
Organization of transcriptional signals in plasmids pBR322 and pACYC184; Stuber D et al.; Electron microscopic analysis of in vitro transcriptional complexes of pBR322 and pACYC184 revealed five and six major transcriptional units, respectively, in these two plasmid vectors . These units are transcribed with various efficiencies, depending upon the individual promoter strengths, which differ in pBR322 up to 10-fold . A most interesting signal arrangement was found at the beginning of the tetracycline resistance region, where two partially overlapping promoters (P1 and P2) initiate transcription crosswise in opposite directions . Whereas P2 is known to promote tetracycline resistance and to be inactivated by HindIII cleavage, P1 is able to transcribe DNA integrated at that site and probably contributes to the expression of the beta-lactamase gene in pBR322 . In pACYC184, besides P1, P2, and the cat (chloramphenicol resistance) promoter (P5), two initiation sites (P3 and P4) were mapped in a region that appears to be part of insertion sequence 1 . The maps of transcription signals permit a more predictable utilization of these cloning vehicles and also allow the reinterpretation of earlier cloning results.

Chemotherapy, 1981, 27(3), 200 - 8
Bacterial elimination and therapeutic effectiveness under different schedules of amoxicillin administration; Klaus U et al.; Bacterial elimination kinetics under different simulated administration schedules of amoxicillin was followed in vitro by using a simple model for simulation of pharmacokinetic parameters . The results were compared to those obtained in lethally infected mice by determining the viable bacterial count in the blood at various times post-infection and under different schedules of amoxicillin administration . A satisfactory correlation could be established between the in vitro and in vivo results . Multiple administration of amoxicillin was found to induce a beta-lactamase in vitro as well as in vivo in the Escherichia coli strain used . No difference was however found between the various administration schedules of amoxicillin in terms of surviving animals . Taking into account the lasting elimination of viable bacteria from the blood, the best treatment of E . coli-infected mice was found to be a twice daily administration of the higher amoxicillin dose.

Mol Gen Genet, 1981, 181(1), 29 - 35
Regulation of plasmid DNA synthesis: isolation and characterization of copy number mutants of miniR6-5 and miniF plasmids; Ely S et al.; Copy number (Cop) mutants of miniR6-5 and miniF plasmid derivatives containing a beta-lactamase gene were isolated by selection for increased ampicillin-resistance . Mutants which exhibited an increased copy number and a reduced incompatibility response as compared to the respective parent mini-plasmid were obtained from both miniR6-5 and miniF . Characterization of these mutant plasmids has provided the first description of the replicative properties of miniF Cop mutants, and has also facilitated a comparison of plasmids representing the IncFI and IncFII incompatibility groups . Cop mutants from these groups differed in several respects: (i) MiniF Cop mutants were considerably more difficult to obtain and showed a markedly lower transforming efficiency than the corresponding miniR6-5 mutants . (ii) MiniR6-5 Cop mutants were stably maintained in a polA1 strain without selective pressure, whereas miniF Cop mutants severely reduced the viability of this host . (iii) MiniR6-5 replication stopped within a few minutes after inhibition of protein synthesis, whereas miniF replication continued at a declining rate for about one hour in the presence of chloramphenicol . (iv) In contrast to miniR6-5 replication, miniF DNA synthesis was blocked faster by rifampicin than by chloramphenicol . (v) The copy number of miniR6-5 plasmids (but not of miniF) was reduced by about 50% in an rnc strain deficient in RNAase III.

Mol Biol Rep, 1980 Dec 31, 6(4), 229 - 34
Effects of prophage Mu induction on expression of adjacent host genes; Dubow MS et al.; The extent of induction and approximate amount of DNA replication of a Mu prophage carrying a gene for ampicillin resistance can be monitored by assaying the level of beta-lactamase . The expression of the lacZ gene adjacent to either end of an induced Mu prophage remains virtually unaffected, until late in the Mu lytic cycle, while Mu DNA is replicating and transposing.

J Med Chem, 1980 Dec, 23(12), 1283 - 92
A multifaceted approach to the study of the side-chain conformation in beta-lactamase-resistant penicillins; Blanpain PC et al.; The resistance of some penicillins to beta-lactamase enzymes was previously attributed to the nature of their C(6) side chain . In order to find explicitly the influence of the conformation of this side chain in the enzymatic mechanism, we have analyzed by experimental and theoretical methods (X-ray diffraction, NMR, IR, PCILO) the molecular structure of six resistant penicillins and derivatives: oxacillin, cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin, cloxacillin sulfoxide, and oxacillinpenicilloic acid . X-ray crystallography of flucloxacillin and nafcillin is fully described . We observe that the side chains of these penicillins have no influence on the electronic properties of the penam nucleus but are much more rigid than in the sensitive ones . The molecular conformations are mostly governed by the nonbonded Van der Waals interactions and, in the oxacillins, partly by the conjugation between exocyclic groups . The lack of flexibility could result in a distorting effect on the structure of the active site of the beta-lactamase, leading to the deactivation of the enzyme.

J Antibiot (Tokyo), 1980 Nov, 33(11), 1256 - 61
Izumenolide-a novel beta-lactamase inhibitor produced by Micromonospora . I . Detection, isolation and characterization; Liu WC et al.; Micromonospora chalcea subsp . izumensis produces a novel beta-lactamase inhibitor izumenolide (EM4615) . Isolation of izumenolide was performed by extraction into butanol under acidic conditions and then back extraction into water at neutrality . The compound was precipitated from the aqueous phase by the addition of calcium or barium salts . Further purification was achieved by distribution in BuOH - 1 N NaOH . Izumenolide is a macrolide containing sulfate ester groups.

Antimicrob Agents Chemother, 1980 Nov, 18(5), 837 - 8
Effect of N-formimidoyl thienamycin (MK0787) on beta-lactamases and activity against beta-lactamase-producing strains; Toda M et al.; N-Formimidoyl thienamycin (MK0787) was active against beta-lactamase-producing strains and was an effective inhibitor of beta-lactamases extracted and purified from these strains except for plasmid-mediated penicillinase type IV.

J Inorg Biochem, 1980 Nov, 13(3), 189 - 204
A spectroscopic study of metal ion and ligand binding to beta-lactamase II; Baldwin GS et al.; beta-Lactamase II has two metal-binding sites . The electronic spectra of Cd(II)- and Co(II)-substituted beta-lactamase II have been investigated . It is suggested that a thiol ligand is involved in metal binding at the first site . The stoichiometric dissociation constants for Co(II) binding to beta-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4 degrees C, 1 M NaCl) by equilibrium dialysis . Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 microM (pH 6.0, 30 degrees C, 1 M NaCl) for the dissociation constant of Zn(II) . The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.

Nature, 1980 Sep 18, 287(5779), 193 - 7
Expression of human fibroblast interferon gene in Escherichia coli; Derynck R et al.; The human fibroblast interferon gene was inserted in a thermoinducible expression plasmid under control of the phage lambda PL promoter . The primary translation products predicted on the basis of the plasmid constructions were hybrid proteins starting with beta-lactamase or phage MS2 polymerase information followed by the total preinterferon . On induction, antiviral activity, whose physico-chemical, immunological and biological characteristics closely corresponded to those of authentic human fibroblast interferon, was synthesized . Processing to a size compatible with mature but unglycosylated authentic product was observed.

Proc Natl Acad Sci U S A, 1980 Sep, 77(9), 5375 - 9
Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule; Shortle D et al.; A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322 . The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction . First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA . Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA . The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite . A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment . Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.

J Bacteriol, 1980 Sep, 143(3), 1127 - 34
Physical mapping and expression of hybrid plasmids carrying chromosomal beta-lactamase genes of Escherichia coli K-12; Grundstrom T et al.; Hybrid plasmids carrying the ampC gene of Escherichia coli K-12 that codes for the chromosomal beta-lactamase were physically studied . The ampC gene was mapped to a deoxyribonucleic acid segment encompassing 1,370 base pairs . The mapping was facilitated by the isolation of a plasmid carrying an insertion of the transposable element gamma delta (gamma delta) close to ampC . The ampA1 mutation, which increases the expression of ampC by a factor of about 20, was localized to a 370-base pair segment of the 1,370-base pair deoxyribonucleic acid segment that contains the ampC gene . Using a minicell protein labeling system, it was seen that plasmids carrying either ampA+, ampC, or ampA1 and ampC coded for a 36,000-dalton protein which comigrated with purified chromosomal beta-lactamase . In cells carrying plasmids that bore the ampA1 allele, the production of this protein was greater . In addition, a protein with a slightly higher molecular weight (38,000) was expressed by both ampA+ ampC and ampA1 ampC plasmids in this protein labeling system . This protein might represent a precursor form of chromosomal beta-lactamasee . From E . coli K-12 strains carrying the ampA1 allele, second-step mutants were isolated that hyperproduced chromosomal beta-lactamase . By reciprocal recombination, plasmid derivatives were isolated that carried these mutations . Two second-step regulatory mutations mapped within the same 370-base pair region as ampA1 . This piece of deoxyribonucleic acid therefore contains ampA, a control sequence region for ampC.

Eur J Biochem, 1980 Aug, 109(1), 97 - 102
Cellular distribution of beta-lactamase RP4 is mediated by an outer membrane protease; Watson DH; RP4 beta-lactamase extracted from the outer membrane of wild-type Escherichia coli can be resolved into several interconvertible forms that differ in their stabilities, substrate profiles and apparent molecular weights . beta-Lactamase isolated from outer membrane of strains which are lacking a protease that is involved in the cleavage of colicins differs from the beta-lactamase of parental cells in substrate profile, apparent molecular weight and the ability to interconvert . The cellular distribution of beta-lactamase also differs between wild-type and protease-deficient mutants . Both strains have equivalent amounts of beta-lactamase in their outer membranes, however the parental strain also has considerable beta-lactamase in the cytoplasmic membrane while the mutant does not . In addition the mutant contains only 30% of the parental level of enzyme in the periplasm . It is proposed that the reduced level of periplasmic enzyme is the result of a defect in processing of membrane-associated beta-lactamase . This conclusion is supported by the observation that the beta-lactamase isolated from the mutant can be converted to forms resembling those found in the parent by incubation with extracts or outer membrane isolated from the parent.

Biochemistry, 1980 Jun 24, 19(13), 2895 - 901
beta-Lactamase proceeds via an acyl-enzyme intermediate . Interaction of the Escherichia coli RTEM enzyme with cefoxitin; Fisher J et al.; The use of cefoxitin, a poor substrate of the RTEM beta-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction . The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly identical with the observed Kcat . Burst kinetics are observed with this substrate, consistent with the rate-limiting deacylation of the cefoxitinoyl-enzyme . That the reaction intermediate involves a covalent link between enzyme and substrate was shown by gel filtration after rapid denaturation of an enzyme-{14C}cefoxitin reaction at the steady state . Fourier transform infrared measurements indicate that the intermediate is an acyl-enzyme involving a hydroxyl group of the beta-lactamase . The evident relationship between the acylation-deacylation sequence of the beta-lactamases and the acylation reaction suffered by the D-Ala-D-Ala-carboxypeptidases is discussed.

J Thorac Cardiovasc Surg, 1980 Jun, 79(6), 933 - 6
Hemophilus influenzae purulent pericarditis in children: diagnostic and therapeutic considerations; Cheatham JE Jr et al.; Purulent pericarditis is an unusual complication of infection in infancy and has been associated with an extremely high mortality rate . Early diagnosis followed by combined antibiotic therapy and surgical drainage of the pericardium has markedly improved survival . Between APril, 1975, and February, 1979, nine patients with purulent pericarditis secondary to Hemophilus influenzae type B were treated at the Oklahoma Children's Memorial Hospital . In every case signs and symptoms of congestive heart failure were present, and a pericardial effusion was demonstrated by echocardiography and confirmed by pericardiocentesis . The organism was identified with countercurrent immunoelectrophoresis and antibiotic sensitivity determined by rapid beta lactamase assay . All patients were treated with a combination of parenteral antibiotics and open surgical drainage of the pericardium . There were no deaths and all patients demonstrated marked improvement following operation . Follow-up echocardiography revealed no evidence of pericardial effusion or signs of constriction in any patient.

Biochem J, 1980 Jun 1, 187(3), 789 - 95
The 1H nuclear-magnetic-resonance spectroscopy of cobalt(II)-beta-lactamase II; Galdes A et al.; The 1H n.m.r . spectra of beta-lactamase II in the presence of Co(II) were studied . Analysis of the spectra suggests that Co(II) binds at the same two metal-binding sites as does Zn(II) . The binding of Co(II) at the first site is much weaker than the binding of Zn(II) at this site, whereas the binding of Co(II) at the second site is tighter than the binding of Zn(II) . The binding of Co(II) to the mono-zinc(II)-enzyme caused only one marked change in the spectrum, namely a decrease in the intensity of the resonances assigned to the C-2 and C-4 protons of one histidine residue (residue E) . However, when the spectra of the apoenzyme and the Co(II)-enzyme were compared, there were many differences . A significant fraction of the protons in the whole molecule are affected by the binding of Co(II) at the first metal-ion-binding site (where the ligands are the enzyme's sole thiol group and three histidine residues) . This may be because the first site is internal, or because of a difference in conformation between the apoenzyme and the mono-Co(II)-enzyme . The second site may be located on the surface of the molecule.

Philos Trans R Soc Lond B Biol Sci, 1980 May 16, 289(1036), 309 - 19
Beta-lactamase inactivation by mechanism-based reagents; Fisher J et al.; The mechanistic pathway followed by the E . coli RTEM beta-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme . There is clear evidence that the beta-lactamase-catalysed hydrolysis of the 7-alpha-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate . An analysis of the inactivation reactions of all the known beta-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a beta-lactamase inactivator . The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N-acyl derivatives of 6-beta-aminopenicillanic acid that are themselves poor substrates for the enzyme . These sulphones are powerful inactivators of the beta-lactamase.

Philos Trans R Soc Lond B Biol Sci, 1980 May 16, 289(1036), 207 - 23
'Beta-lactams' as beta-lactamase inhibitors; Cole M; The application of inhibitors to block the beta-lactamase destruction of penicillins and cephalosporins by resistant bacteria is a potentially useful way of improving the efficacy of established compounds . Certain semi-synthetic penicillins and cephalosporins have been found to be competitive inhibitors of selected beta-lactamases but an examination of streptomycete culture fluids has revealed two new types of beta-lactam compound: clavulanic acid, which is a progressive inactivator of a wide range of beta-lactamases, and the olivanic acids, which are both broad-spectrum antibiotics and potent beta-lactamase inhibitors . Penicillanic acid sulphone and 6-beta-bromopenicillanic acid have been shown to be significant inhibitors of beta-lactamase . The chemotherapeutic application of these compounds is discussed.

Nucleic Acids Res, 1980 May 10, 8(9), 2055 - 65
Synthesis of part of a mouse immunoglobulin light chain in a bacterial clone; Amster O et al.; We have cloned double stranded cDNA sequences encoding a mouse immunoglobulin light chain (L-321) into the PstI site of the beta-lactamase gene of plasmid pBR322 by the oligo (dG)-oligo (dC) tailing procedure . Escherichia coli X1776 transformed by the recombinant plasmids were screened for the expression of L-321 antigenic determinants by a newly developed in situ radio-immunoassay . One out of seven transformants screened was found to synthesize an L-chain like protein . Each bacterial cell produces about 550 molecules of the L-chain sequence . Preferential segregation of the L-chain sequence to the periplasmic space suggest covalent attachment of the L-chain sequence to the N-terminal portion of beta-lactamase . Restriction mapping of the plasmid DNA isolated from the positive clone indicated the presence of a DNA sequence coding for the entire constant region and extending into the variable region for a length corresponding to about 40 amino acid residues . The orientation of the cloned cDNA with respect to the plasmid DNA is compatible with the formation of a fused beta-lactamase-L-321 peptide.

J Bacteriol, 1980 May, 142(2), 668 - 82
Intramolecular transposition and inversion in plasmid R6K; Chiang SJ et al.; Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance . Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660 . A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid . A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1 . pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain . Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon . All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid . In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred . These results are discussed in terms of current models of transposition.

Proc Natl Acad Sci U S A, 1980 May, 77(5), 2450 - 4
Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H; Itoh T et al.; A plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid ColE1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed . The plasmid DNA has three principal sites where transcription is initiated in vitro . One is located in the ColE1 segment 555 nucleotides upstream from the origin . Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions . Cleavage of these transcripts by RNase H generates 3' termini at the origin region . When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus . The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColE1 DNA synthesis . Once it has served as a primer, the RNA is eliminated from the product by RNase H.

J Antibiot (Tokyo), 1980 Apr, 33(4), 420 - 5
Multiple effects induced by unstable mutation in Streptomyces lavendulae; Nakano MM et al.; Arg mutants, isolated from Streptomyces lavendulae at unusually high frequencies, showed several phenotypic characteristics . The characteristics common to all arg mutants include: (1) repression of beta-lactamase production, (2) inhibition of aerial mycelium formation, (3) development of acid pH, (4) low saturation density of growth in liquid culture, (5) a decrease in antibiotic production, (6) an increase in sensitivity to benzylpenicillin and (7) a decrease in production of pigment . These results suggest that the arg mutation concomitantly caused the depression of secondary metabolism in S . lavendulae.

Proc Natl Acad Sci U S A, 1980 Mar, 77(3), 1570 - 4
Introduction of transposon Tn901 into a plasmid of Anacystis nidulans: preparation for cloning in cyanobacteria; van den Hondel CA et al.; We have used the TEM beta-lactamase transposon Tn901, located on Escherichia coli plasmid pRI46, to introduce in vivo a genetic marker into plasmid pUH24, present in the cyanobacterial strain Anacystis nidulans R-2 . Restriction enzyme analysis and heteroduplex studies of the 8.3 x 10(6)-dalton plasmids pCH1-pCH5, present in the ampicillin-resistant A . nidulans R-2 colonies obtained after transformation with pRI46, demonstrated that these plasmids consist of the complete sequence of Tn901 inserted at different places into plasmid pUH24 . The pUH24::Tn901 recombinant plasmids transform A . nidulans R-2 with a frequency of 10(-4)--10(-5) per microgram of plasmid DNA and contain a single cleavage site for the restriction enzyme Xho I . From pCH1 a plasmid of 5.5 x 10(6) daltons,pUC1, was constructed with only a part of the Tn901 sequence and an additional single cleavage site for the restriction enzyme BamHI . This plasmid, as well as plasmids pCH1-pCH5, are potentially useful as vectors for cloning genes in cyanobacteria and for studying cyanobacterial plasmid biology.

Antimicrob Agents Chemother, 1980 Feb, 17(2), 124 - 8
Unstable mutation of beta-lactamase production in Streptomyces lavendulae; Matsubara-Nakano M et al.; Streptomyces lavendulae S55-B1 gave two distinct variants at an unusual high frequency: one is a beta-lactamase-nonproducing variant and the other is an Arg- variant . All of the Arg- variants concomitantly had no, or only very low, beta-lactamase activity and were unable to form aerial mycelia or spores . There was no significant linkage between the beta-lactamase activity and the other nutritional requirement which was analyzed . Two of the Arg- variants spontaneously gave Arg+ revertants at a very low frequency . The revertants, however, did not recover the beta-lactamase activity . It is suggested that the beta-lactamase gene may be capable of transposition with inactivation of the arg gene occurring frequently by insertion of the transposed element . Covalently closed circular deoxyribonucleic acid from S55-B1 was not detected by either cesium chloride-ethidium bromide buoyant density centrifugation or agarose gel electrophoresis.

Antimicrob Agents Chemother, 1980 Jan, 17(1), 16 - 9
Nuclear magnetic resonance spectrometric assay of beta-lactamase; Kono M et al.; Beta-Lactam antibiotics and the crude enzyme were mixed in deuterium oxide and placed in a nuclear magnetic resonance tube . The change of the nuclear magnetic resonance spectrum during the enzymatic reaction was then analyzed to determine beta-lactamase activity . By using beta-lactam antibiotics such as penicillins, cephalosporins, and cephamycins as substrates, a comparison of the beta-lactamase activities was made between the nuclear magnetic resonance spectrometric assay and the iodometric assay . There was a close correlation between these two methods.

Mol Gen Genet, 1980, 180(2), 249 - 57
Tandem duplication induced by an unusual ampA1-, ampC-transducing lambda phage: a probe to initiate gene amplification; Edlund T et al.; Secondary attachment site lambda-lysogens were isolated in an Escherichia coli strain carrying multiple tandem 9.8 kb repeats . The repeat carried the structural gene for chromosomal beta-lactamase, ampC . One lysogen produced lysates with amp-transducing activity . Three types of phages with different densities were obtained from this lysogen . The one with the lowest density was found to be a helper lambda cI857S7 phage . The other two phage showed identical restriction endonuclease fragmentation patterns . The difference in density was due to the presence or absence of phage tail . In lambda damp the right cohesive end segment was deleted in a random fashion with the majority ending between 81.0% and 82.4% of lambda . The chromosomal segment of lambda damp was most likely located at the lambda attachment site . The lambda damp DNA was compared to that of ColE1 hybrid carrying the chromosomal amp segment and a ColE1 hybrid carrying the same 9.8 kb amp repeat as the lysogen from which lambda damp was isolated . It was found that the chromosomal part of lambda damp constituted 9.8 kb, i.e . the size of one repeat . Moreover, the novel joint between adjacent repeats was present . In a lambda attB-deleted E . coli K-12 strain, lysogenic for lambda damp, highly ampicillin-resistant mutants occurred at an exceedingly high frequency . They were found to contain in the chromosome an amplified 9.8 kb repeat . This suggested that integration of the novel joint for lambda damp into the amp region gives rise to an amplifiable duplication . In E . coli lysogenized for lambda damp at lambda attB highly ampicillin-resistant clones were also found at a high frequency . These clones carried multiple tandem repeats of lambda damp DNA, each with an intact right end segment.

Antimicrob Agents Chemother, 1979 Dec, 16(6), 772 - 5
Increased production of beta-lactamase under anaerobic conditions in some strains of Escherichia coli; Rashtchian A et al.; A simple biological assay to detect beta-lactamase activity exhibited by selected cultures of Escherichia coli was used to test enzyme production in cells incubated aerobically and anerobically . Anaerobic incubation resulted in increased size of zones of drug inactivation by some beta-lactamase-producing strains . The beta-lactamase activity of cell lysates was determined iodometrically for aerobically and anaerobically grown cells . The specific beta-lactamase activity for anaerobically grown cells was three to five times greater than for aerobically grown cells . Beta-lactamase production was determined to be constitutive in all strains and to be plasmid mediated, as demonstrated by transfer to E . coli K-12 by conjugation.

Eur J Biochem, 1979 Dec, 102(1), 65 - 71
Production of a soluble form of fumarate reductase by multiple gene duplication in Escherichia coli K12; Cole ST et al.; 1 . Ampicillin-hyperresistant mutants of Escherichia coli K12 bearing multiple gene duplications in the ampC (beta-lactamase) gene region of the chromosome overproduced at least six proteins with molecular weights 97,000, 80,000, 72,000, 49,000, 33,000 and 26,500 during anaerobic growth . All but two of the proteins (80,000-Mr and 49,000-Mr) were also overproduced during aerobic growth . The distribution of the proteins in soluble and particulate cell fractions was investigated . 2 . The 33,000-Mr and 72,000-Mr components were identified as beta-lactamase and the amp-linked frdA gene product, fumarate reductase, respectively . Co-sedimentation of the 26,500-Mr component with the fumarate reductase suggested that the smaller protein could be functionally related to the reductase . The lack of correspondence between the amplified proteins and the products of other amp-linked genes, aspA and mop(groE), indicated that these genes are not included in the repetitive sequence . 3 . Fumarate reductase activities were amplified up to 32-fold by the multiple gene duplications . Two forms of fumarate reductase were produced: particulate (membrane-bound) and soluble (cytoplasmic) . Production of the soluble form occurred when the binding capacity of the membrane was saturated . Both forms of fumarate reductase were enzymically active but the soluble form was readily inactivated under assay conditions.

Cell, 1979 Dec, 18(4), 1153 - 63
DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3; Heffron F et al.; The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported . The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site . By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map . This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity . These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase . The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose . Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA . We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978) . A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.

J Antibiot (Tokyo), 1979 Dec, 32(12), 1328 - 35
Purification of beta-lactamase from Streptomyces cellulosae by affinity chromatography on Blue Sepharose; Ogawara H et al.; A beta-lactamase from culture supernatant of Streptomyces cellulosae was purified about 1,450-fold to apparent homogeneity in polyacrylamide gel electrophoresis and isoelectric focusing on polyacrylamide gel sheet . The methods used were ammonium sulfate precipitation, CM-52 cellulose ion-exchange chromatography and affinity chromatography on Blue Sepharose CL-6B . The molecular weight was determined to be approximately 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This value was in good agreement with the previous value determined by gel filtration on Sephadex G-75 . The isoelectric point was pH 9.5 . The enzyme behaved primarily as penicillinase and apparent Km value for benzylpenicillin was 500 microM . The beta-lactamase of S . cellulosae interacted strongly with blue dextran and NADP+-agarose but not with Sepharose . In addition, the presence of NADP+ but not NAD+ and ATP diminished sharply the intrinsic fluorescence intensity of the enzyme and the apparent association constant was calculated to be 1.4 x 10(3) M-1 . The beta-lactamase decreases its enzymatic activity against benzylpenicillin in the presence of NADP+ . From these results, it is suggested that this beta-lactamase has a dinucleotide binding fold.

Ann Microbiol (Paris), 1979 Oct, 130B(3), 295 - 304
{"Beta-lactamase enzymogram": an agar adaptation of the iodometric method (author's transl)}; Labia R et al.; This paper deals with an agar-gel adaptation of the Perret-Novick iodometric titration of degradation products from beta-lactam antibiotics when they are submitted to a beta-lactamase action . Petri dishes containing an agar-gel make up from the classical iodine-iodide-starch system, initially present a dark blue colour.When beta-lactam compounds are opened by beta-lactamases from crude extracts or whole bacteria, the gel is locally destained . Two experimental variants of this procedure are proposed . This semi-quantitative, inexpensive, accurate and simple method is very convenient for routine experiments . Thus with crude extracts or even whole cells, information can be obtained about beta-lactamase specific activities, substrate profils or activity of inhibitors such as cloxacillin or clavulanic acid.

Mol Gen Genet, 1979 Oct 1, 175(3), 239 - 43
The nature of the genetic determinant for the SHV-1 beta-lactamase; Nugent ME et al.; In two unrelated plasmids of incompatibility groups FII and N the gene for the SHV-1 beta-lactamase exists as part of a transposable element of molecular weight 9.5 megadaltons . This transposon has moved onto plasmids of at least three incompatibility groups; PI, Ialpha and J . This confirms the suggestion that the recent spread of the SHV-1 beta-lactamase has been associated with the transposition of the genetic determinant of this enzyme between unrelated plasmids.

Proc Natl Acad Sci U S A, 1979 Oct, 76(10), 4837 - 41
Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells; Achtman M et al.; Most tra proteins encoded by the Escherichia coli F sex factor are incorporated into the minicell envelope . We have now assigned the tra proteins to cytoplasm (TraIp and 2b), inner membrane (TraEp, TraMp, and TraSp), and outer membrane (6e, TraAp, TraBp, TraJp, TraKp, TraLp, and TraTp) . two proteins, TraDp and 6d, were associated with both inner and outer membranes . The proteins exported to the inner or outer membranes did not undergo proteolytic cleavage (processing) whereas beta-lactamase was processed normally.

Methods Find Exp Clin Pharmacol, 1979 Oct, 1(4), 233 - 8
Factors influencing the inactivation of aminoglycosides by beta-lactams; Flournoy DJ; Aminoglycosides and beta-lactam antibiotics were tested alone and in combination, via bioassay, in the presence of pH buffers at 6.5, 7.5 and 8.5; human serum; MgCl2 and CaCl2; clavulanic acid and beta-lactamase . Results show: inactivation of gentamicin, tobramycin, sisomicin and netilmicin is influenced by pH; clavulanic acid can inactivate gentamicin and tobramycin; and inactivation is influenced by salt concentration and constituents.

Gene, 1979 Oct, 7(2), 121 - 39
Synthesis of proteins coded by plasmid vectors of pCV series (Apr, Tcr) and their recombinant derivatives (pDm) in E . coli minicells; Kopylova-Sviridova TN et al.; Polypeptide synthesis directed by vector plasmids of pCV series conferring ampicillin and tetracycline resistance (Apr, Tcr) and by recombinant plasmids (pDm) have been analyzed using the minicell system . It has been found that a polypeptide of 34 000 daltons is responsible for the Tcr phenotype and regulated from the promoter near the HindIII site . Cloning of DNA fragments into HindIII site allowed to conclude that DNA from Drosophila melanogaster contains nucleotide sequences which may act as promoters for a 34 000 dalton polypeptide gene . beta-Lactamase is expressed as five proteins of 24 000, 26 5000, 27 000, 28 500 and 29 500 daltons . Insertion of DNA fragments into PstI site prevents the synthesis of all five polypeptides . Recombinant clones Dm39 and Dm187 produce additional proteins of 19 000, 23 000, 24 000 and 27 000 daltons.

Mol Gen Genet, 1979 Jun 7, 173(2), 115 - 25
Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12; Edlund T et al.; A ColEl hybrid plasmid, pNUl, carrying the amp operon coding for chromosomal beta-lactamase was isolated from the Clarke and Carbon collection and physically mapped . The physical location of ampC within this plasmid was further deduced by in vitro cloning . By reciprocal recombination between pNUl and chromosome of two unstable beta-lactamase hyperproducing E . coli K-12 mutants a large plasmid from each mutant was obtained . The respective plasmid was physically mapped and found to contain five and two repeated DNA segments . The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem . The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene . The chromosomal DNA of the beta-lactamase hyperproducing E . coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid . The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E . coli.

Gene, 1979 Jun, 6(2), 91 - 106
Plasmids with temperature-dependent copy number for amplification of cloned genes and their products; Uhlin BE et al.; Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1 . At 30 degrees C these miniplasmids are present in 20--50 copies per cell of Escherichia coli, whereas at temperatures above 35 degrees C the plasmids replicate without copy number control during 2--3 h . At the end of this period plasmid DNA amounts to about 75% of the total DNA . During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products . Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plamid pKN410 has a single BamHI site and carries ampicillin resistance . The plasmids can therefore be used as cloning vectors . Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded beta-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication . Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products . These plasmids have lost their mobilization capacity . Runaway replication is lethal to the host bacteria in rich media . These two properties contribute to the safe use of the plasmids as cloning vehicles.

J Bacteriol, 1979 Jun, 138(3), 896 - 902
In vivo regulation of chromosomal beta-lactamase in Escherichia coli; Jaurin B et al.; Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo . Both the activity and the amount of beta-lactamase increased with growth rate . During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation . After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min . To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature . No influence by the relA gene product was found on beta-lactamase synthesis . The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.

J Bacteriol, 1979 Apr, 138(1), 48 - 54
Polypeptides expressed in Escherichia coli K-12 minicells by transposition elements Tn1 and Tn3; Dougan G et al.; Escherichia coli K-12 minicells were employed to examine polypeptides encoded by plasmids carrying wild-type and mutant Tn1 or Tn3 transposition elements . Tn1- and Tn3-containing minicells express high levels of four transposon-specified polypeptides . Three, of molecular weights 30,000, 28,000, and 25,000, are related immunologically to beta-lactamase, the enzyme responsible for ampicillin hydrolysis . A fourth polypeptide of molecular weight 19,000 is encoded by the Tn1 or Tn3 region which spans the BamHI cleavage site . Mutant transposons which no longer produce this polypeptide transpose at higher than wild-type frequencies to give aberrant transposition products (Gill et al., J . Bacteriol . 136: 742--756, 1978; Heffron et al., Proc . Natl . Acad . Sci U.S.A . 72:3632--3627, 1975) . No expression could be detected from a region of the transposons extending from the inverted repeat sequence distal to the beta-lactamase gene to more than half the distance into the Tn1 or Tn3 sequence.

Br J Surg, 1979 Mar, 66(3), 188 - 90
Randomized controlled trial of cefuroxime for established postoperative respiratory infection; Litchfield JC et al.; A randomized controlled trial has investigated the value of cefuroxime (a new antibiotic resistant to beta-lactamase) in 80 patients with established postoperative respiratory infection . Although the majority of respiratory isolates were sensitive to cefuroxime, there was no significant advantage in the antibiotic group compared with the controls with respect to duration of fever, radiological abnormality or infected sputum . We conclude that antibiotics are rarely necessary for the majority of patients who develop postoperative respiratory infections.

Infect Immun, 1979 Mar, 23(3), 799 - 810
Studies on gonococcus infection . XVIII . 125I-labeled peptide mapping of the major protein of the gonococcal cell wall outer membrane; Swanson J; The major outer membrane proteins from 10 gonococcal strains were examined after 125I-labeling of the proteins as single bands resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate . These 125I-proteins were then treated with either trypsin or alpha-chymotrypsin, and the resultant 125I-peptides were visualized by autoradiography after two-dimensional electrophoretic and chromatographic separation on thin-layer cellulose sheets . Several 125I-peptides were present in all the major outer membrane proteins examined . The presence and absence of additional 125I-peptides segregated the major proteins into two pattern groups . One group consisted of major outer membranes with molecular weights of 34,000 or 33,000; major proteins with molecular weights of 32,000 constituted the other group . Two beta-lactamase-producing gonococcal isolates were examined . Their major outer membrane proteins were identical in apparent molecular weights and alpha-chymotryptic 125I-peptide fingerprints; these proteins contained 125I-peptides not found in other gonococcal major proteins . No 125I-peptide differences were found among the major outer membrane proteins of strain F62 gonococci that exhibited differences in piliation and/or colony opacity characteristics.

J Bacteriol, 1979 Feb, 137(2), 990 - 9
Location of an ampicillin resistance transposon, Tn1701, in a group of small, nontransferring plasmids; Yamada Y et al.; By restriction endonuclease cleavage mapping and electron microscopic examination of heteroduplexes, we have identified an ampicillin resistance determinant transposon, designated Tn1701, in a group of small, nontransferring plasmids which confer resistance to ampicillin (Ap), sulfonamide (Su), and streptomycin (Sm) . Plasmid NTP1, which mediates Ap resistance, contains Tn1701 . Recombinant plasmids NTP3 (Ap Su) and NTP4 (Ap Su Sm) contain Tn1701, indicating that they were derived by transposition of Tn1701 from NTP1 to an unrelated plasmid, NTP2 (Su Sm) . The transposon Tn1701 is very similar to the known ampicillin resistance transposons Tn1, Tn2, and Tn3 in its size (3.2 x 10(6) daltons), base sequence homology observed by heteroduplex formation, restriction endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends . Like the other TnA elements, Tn1701 also specifies a type TEM beta-lactamase.

Mol Gen Genet, 1979, 177(1), 13 - 22
Construction and characterization of a plasmid coding for a fragment of the Escherichia coli recA protein; Little JW; The E . coli recA gene was cloned from the phage lambda precA into the vector pBR313 . A plasmid, pJL3, was also isolated by cloning a portion of the recA gene into the vector pBR322 . pJL3 coded for a fragment of the recA protein 34 Kd (kilodaltons) in size (compared to 40 Kd for the intact protein) . This fragment was antigenically related to the recA protein and its synthesis was subject to the same controls as that of the recA protein . The fragment did not express any detectable recA function . When wild-type cells with pJL3 were treated with nalidixic acid, the 34 Kd fragment and the beta-lactamase, made from a gene located downstream from the recA segment, were expressed at very high levels . Moreover, in these cells the rate of synthesis of intact recA protein from the chromosome was inhibited about 2-fold, relative to other chromosomal proteins, when compared to wild-type cells with the pBR322 vector . High level expression of the recA protein fragment and/or the beta-lactamase appeared to be lethal . The size of the 34 Kd fragment, taken together with the location of chain-termination codons in pJL3, localizes the regulatory region of the recA gene within 100 base pairs.

Biochem J, 1979 Jan 1, 177(1), 365 - 7
6 beta-Bromopenicillanic acid inactivates beta-lactamase I; Knott-Hunziker V et al.; The inactivation of beta-lactamase I by preparations of 6alpha-bromopenicillanic acid showed unexpected kinetic features that indicated that the active species was the 6beta-epimer . Samples containing 6beta-bromopenicillanic acid have been synthesized and shown to inactivate the enzyme in a rapid stoicheiometric reaction.

Nature, 1978 Dec 21-28, 276(5690), 795 - 8
Synthesis of growth hormone by bacteria; Seeburg PH et al.; A hybrid gene was constructed between the beta-lactamase gene of plasmid pBR322 and the cloned coding sequence for rat growth hormone . This gene is expressed in bacteria and growth hormone sequences are detectable by immunological methods.

Proc Natl Acad Sci U S A, 1978 Dec, 75(12), 6012 - 6
In vitro mutagenesis of a circular DNA molecule by using synthetic restriction sites; Heffron F et al.; A method for mutagenizing circular DNA molecules has been developed that uses synthetic oligodeoxynucleotide restriction sites as mutagens . A single synthetic restriction site is introduced at random by cleaving circular DNA with a nonspecific double-strand endonuclease . The restriction site is then ligated to the ends and the molecule is subsequently recircularized . These small additions to the genome are mapped by digestion with the appropriate restriction enzyme . Rearrangements such as duplications and deletions can be engineered at will by using the added restriction sites . This technique has been used to produce a fine-structure map of RSF1050, a ColE1 derivative, 60% of which is a transposable DNA sequence encoding the TEM beta-lactamase (Tn3) . A subset of the mutations, mapping within a narrow region of Tn3, result in an increased frequency of Tn3 transposition; mutations in other regions abolish transposition entirely.

J Bacteriol, 1978 Aug, 135(2), 612 - 21
Isolation and characterization of a Clo DF13::Tn901 plasmid mutant with thermosensitive control of DNA replication; Andreoli PM et al.; After nitrosoguanidine mutagenesis, a mutant Escherichia coli strain harboring the Clo DF13::Tn901 plasmid pJN03 was isolated that is thermosensitive (Ts) for growth at 43 degrees C . The mutation responsible for this thermosensitive phenotype resides on the pJN03 plasmid genome . Cells harboring the pJN03 cop-1(Ts) plasmid mutant showed a large increase in plasmid copy number at 43 degrees C accompanied by an increase in the synthesis of plasmid-specified gene products like cloacin DF13 and beta-lactamase . The pJN03 cop-1(Ts) mutant showed uncontrolled plasmid DNA replication at the nonpermissive temperature . Analysis of plasmid deletions showed that the mutation is located in the Clo DF13 map interval from 0 to 12% or 29 to 45% . This implies that native cloacin DF13 and the Clo DF13-specified polypeptides B, C, D, E, and G are not involved in the pleiotropic phenotype of the plasmid mutant pJN03 cop-1(Ts).

Br J Vener Dis, 1978 Jun, 54(3), 165 - 7
The efficacy of cefuroxime for the treatment of acute gonorrhoea in men; Price JD et al.; This is a report on a clinical trial in which cefuroxime was used for treating 110 men with uncomplicated urethral gonorrhoea . Twenty-three men were given 1 g cefuroxime intramuscularly and 1 g probenecid orally, and 87 were treated with 1.5 g cefuroxime intramuscularly with 1 g probenecid orally . All 18 patients treated with 1 g cefuroxime and 1 g probenecid and seen at least once after treatment, were cured . Sixty-six (98.5%) of 67 patients treated with 1.5 g cefuroxime and 1 g probenecid and seen at least once after treatment, were cured . No side effects were reported . The high cure rates compared favourably with other antibiotics now in regular use and cefuroxime should be of great value especially for patients infected with beta-lactamase producing gonococci.

Biochemistry, 1978 May 30, 17(11), 2185 - 9
Chemical studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid; Charnas RL et al.; Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme . Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity . Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm . Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme . Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme . It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.

Biochemistry, 1978 May 30, 17(11), 2180 - 4
Kinetic studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid; Fisher J et al.; The kinetic details of the irreversible inactivation of the Escherichia coli RTEM beta-lactamase by clavulanic acid have been elucidated . Clavulanate is destroyed by the enzyme and simultaneously inhibits it by producing two catalytically inactive forms . One of these is transiently stable and decomposes to free enzyme (k = 3.8 X 10(-3) S-1), while the other corresponds to an irreversibly inactivated form . The transient complex is formed from the Michaelis complex at a rate (k approximately 3 X 10(-2) S-1) which is some threefold faster than the rate of formation of the irreversibly inactivated complex . The transient complex is, therefore, the principle enzyme form present after short time periods . In the presence of excess clavulanate, however, all the enzyme accumulates into the irreversibly inactivated form . The number of clavulanate turnovers that occur prior to complete enzyme inactivation is 115.

Mol Gen Genet, 1978 Mar 20, 160(1), 1 - 11
Genetic map of the bacteriocinogenic plasmid CLO DF13 derived by insertion of the transposon Tn901; Andreoli PM et al.; An ampicillin transposon Tn901 was used as a "mutagen" to isolate insertion mutants of the bacteriocinogenic plasmid Clo DF13 . By combining the obtained heteroduplex and restriction maps of the Clo DF13::Tn901 plasmids (van Emboden et al., 1977b) with their polypeptide pattern in minicells, we were able to map five genes on the Clo DF13 genome . These five genes designated A (cloacin gene), B, C, D, and G cover 55% of the coding capacity of Clo DF13 DNA . Since integration of Tn901 within these five genes did not result in a loss of the Clo DF13::Tn901 plasmids involved, it is suggested that these genes do not play an essential role in the maintenance of these plasmid insertion mutants . In addition, the described methods allowed us to indicate the initiation site of cloacin synthesis and to propose the counter-clockwise direction of transcription of the cloacin gene . The Tn901 DNA directed the synthesis of at least three polypeptides one of which is shown to be a TEM-1 beta-lactamase.

Arzneimittelforschung, 1978, 28(8), 1327 - 30
Biophotometric investigations on the influence of cephradine and related cephalosporins on the growth of beta-lactamase-producing staphylococi; Breuer A et al.; Biophotometric growth studies were conducted with clinical isolates of beta-lactamase-producing staphylococi in the presence and absence of cephradine, cephalexin, cefazolin and cephacetrile . Of the four antibiotics, cephradine was the most effective in prolonging the suppression of bacterial growth resulting from antibiotic action while cefazolin was the least effective . Cephalexin and cephacetrile were intermediate in effect . The superiority of cephradine over cephalexin was shown to be related to the more rapid destruction of cephalexin by beta-lactamase . On comparing the results of these studies with clinical experience it was concluded that biophotometric studies have clinical relevance and consequently should be used for evaluating the efficacy of beta-lactam antibiotics against pathogenic staphylococi.

Infection, 1978, 6(5), 242 - 3
{Beta-lactamase producing gonococci in Munich (author's transl)}; Milatovic D et al.; Two more penicillinase-producing gonococci strains have been isolated in Munich . The source of infection could not be traced in a 20 year old prostitute . A 45 year old patient contracted the infection during a stay in Bangkok.

J Bacteriol, 1977 Oct, 132(1), 1 - 7
Resistance of Escherichia coli to penicillins: fine-structure mapping and dominance of chromosomal beta-lactamase mutations; Normark S et al.; Seven Escherichia coli K-12 mutants with a lowered chromosomal beta-lactamase activity were analyzed genetically . The beta-lactamase-negative mutants isolated from ampA1-carrying strains (resistant to 10 microgram of ampicillin per ml) all carried genetic lesions very close to the ampA1 mutation, which was still present . In an earlier report, two of the mutations mediating a beta-lactamase-negative phenotype (L . G . Burman, T . Park, E . B . Linstrom, and H . G . Boman, J . Bacteriol . 116:123-130, 1973) were shown to have occurred in the structural gene for beta-lactamase, designated ampC . It is suggested that all beta-lactamase-negative mutants studied here were altered in ampC . The relative order of ampC mutations was (ampC1, ampC8)-ampC9-(ampC12, ampC14)-ampC11, and the gene order was found to be ampC-1mpA-purA . The ampA1 allele was dominant over its wild-type allele but acted only cis and not trans, suggesting that ampA is the promoter or operator region for ampC . A gene dosage effect was found for strains homozygous for ampA+ ampC+ or ampA1 ampC+ . Heterozygotes carrying the ampC8 allele on the chromosome showed an apparent derepression of the episomal ampC allele, suggesting a role for beta-lactamase in its own regulation.

C R Acad Sci Hebd Seances Acad Sci D, 1977 May 2, 284(17), 1729 - 32
{Problems in the determination of isoelectric points of beta lactamases}; Labia R et al.; Analytical isoelectric focusing in polyacrylamide gels and preparative electrofocusing in density gradients may give very different pI determination for the same beta lactamase . This difference seems due to a lack of enzyme migration in some parts of the cross-linked polyacrylamide gel . This migration appears easier in density gradient isoelectricfocusing, and thus this technique yields more significant absolute pI values . Analytical isoelectric focusing should only be used for comparison studies.

J Bacteriol, 1977 May, 130(2), 852 - 9
Synthesis of R-plasmid-coded beta-lactamase in minicells and in an in vitro system; Grindley JN et al.; beta-Lactamase encoded by a small, nontransferring R-plasmid, NTP1, conferring ampicillin resistance to its host bacteria, was purified . NTP1 plasmid-coded beta-lactamase was found to be periplasmically located in the host Escherichia coli cell, to have a molecular weight of about 25,000, and to show a relatively low activity against oxacillin and methicillin compared with benzylpenicillin . These characteristics indicate that NTP1 plasmid-coded beta-lactamase is very similar or identical to the "TEM-type" beta-lactamase, which is the most common beta-lactamase coded by R-plasmids in enteric bacteria . In minicells containing NTP1 plasmids, at least six plasmid-specific proteins were synthesized, and beta-lactamase was synthesized in a greater amount than other plasmid-coded proteins . In a cell-free transcription-translation coupled system from E . coli, NTP1 plasmid deoxyribonucleic acid directed the synthesis of several species of plasmid-specific proteins, including active beta-lactamase . The in vitro system also showed preferential synthesis of beta-lactamase, as was observed in minicells containing NTP1 plasmids.

Gene, 1977 May, 1(3-4), 241 - 53
Properties of a transposon conferring resistance to penicillins and streptomycin; Hedges RW et al.; R938 carries a transposon (TAbeta) of approximate molecular weight 9.5 Megadaltons (Mdal, 10(6) daltons) . This contains genes for a beta lactamase of type TEM-1 and for streptomycin phosphatransferase (SPT) . There is a ten-fold difference in the efficiency of transposition in different strains of E . coli K12.

Mol Gen Genet, 1977 Mar 7, 151(2), 151 - 60
The transposon Tn1 as a probe for studying ColE1 structure and function; Dougan G et al.; Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutnat phenotypes . Whereas all plasmid examined were present in normal amount, all showed reduced immunity to killing by colicin E1 . Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome . The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation . Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids . The other two are nontransmissible and produce colicin . Non-transmissibility is correlated with reduced relaxation complex . Patterns of protein synthesis in minicells by ColE1 and ColE1 :: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be beta-lactamase . ColE1 :: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.

Ital J Biochem, 1977 Jan-Feb, 26(1), 37 - 43
The inactivation of beta-lactamase I from B . Cereus by dicloxacillin: effect of buffer composition on the kinetic characteristics of the modified enzyme; Salifi V et al.; Buffer composition affects the pattern of inactivation of B . cereus beta-lactamase I by dicloxacillin . At pH 7.0, in the presence of phosphate ions, dicloxacillin appears to cause essentially an irreversible inactivation of the catalytic site of the enzyme, while in a tris-cacodylate buffer the inactivation process appears to affect essentially the substrate-binding site . Buffer ions act presumably by interacting themselves with either the catalytic or the substrate-binding site of the enzyme, thus modifying the affinity of these sites for the halogenated isoxazolyl-penicillins.

J Bacteriol, 1976 Oct, 128(1), 425 - 34
Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion; Rubens C et al.; Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010 . There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells . The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases . Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations . Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar . In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number.

Mol Gen Genet, 1976 Apr 23, 145(1), 101 - 8
Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids; Humphreys GO et al.; An investigation of in vitro mutagenesis of plasmid DNA with hydroxylamine is described . The treated plasmid DNA was used to transform Escherichia coli K12 . Mutants of the plasmid NTP3, which codes for resistance to ampicillin and sulphonamides, were isolated and characterised . They were classified according to the reduction in level of their beta-lactamase activity . Hydroxylamine-induced mutants of NTP14 were also isolated . This plasmid codes for ampicillin resistance, synthesis of colicin E1, and the EcoRI restriction and modification enzymes . One class of mutants is lethal to the host strain at temperatures above 33 degrees C, but carrier strains grow well at 28 degrees C . There is evidence that these mutants code for a temperature-sensitive EcoRI modification activity: the lethal effect probably results from the cleavage of the host-cell DNA by the restriction enzyme at non-permissive temperatures . The possible genetic uses of the mutant plasmids for the production of hybrid plasmids in the bacterial cell are discussed.

Biochem J, 1975 Sep, 149(3), 547 - 51
The pH-dependence and group modification of beta-lactamase I; Waley SG; The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied . Benzylpenicillin and ampicillin (6-{D(-)-alpha-aminophenylacetamido}penicillanic acid) were used . Both kcat . and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH . The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin) . For benzylpenicillin both kcat . and kcat./Km depended on pH in exactly the same way . The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate . The pH-dependence of kcat . for ampicillin differed, however, presumably because of the polar group in the side chain . The hypothesis that the pK5 group is a carboxyl group was tested . Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate . These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.

J Bacteriol, 1975 Jul, 123(1), 179 - 86
Replication of R-factor R1 in Scherichia coli K-12 at different growth rates; Engberg B et al.; The R-factor R1drd-19 mediates resistance to beta-lactam antibiotics via a beta-lactamase . A strain of Escherichia coli K-12 carrying R1drd-19 was grown at different growth rates by using different carbon sources . The specific rate of production of the R1 beta-lactamase increased linearly with the growth rate and with the gene dosage . The content of R1 deoxyribonucleic acid was estimated by alkaline sucrose gradient centrifugation and by analysis of the specific rate of beta-lactamase synthesis in nutritional shift-up experiments and was found to decrease fivefold when the growth rate was increased from 0.4 to 1.8 doublings per h . The number of R1 molecules per cell decreased from six to two in the same growth range . The presence of the plasmid affected the mean cell size significantly; at a growth rate of 0.4 doublings per h the R-+ cells were on the average 50% bigger than the R-minus cells, whereas the effect was less than 10% at a growth rate of 1.8 doublings per h . Several reports in the leterature state that the initiation mass of chromosome replication is constant . In this paper it is shown that the initiation mass of R1 replication is proportional to the growth rate . Thus, the replication of the plasmid R1 and of the chromosome are independently regulated processes . It is argued that plasmid replication is under negative control.

J Bacteriol, 1975 Apr, 122(1), 250 - 6
Origin of the TEM-beta-lactamase gene found on plasmids; Heffron F et al.; A sequence of deoxyribonucleic acid of 2.7 times 10-6 to 3.3 times 10-6 daltons which includes the TEM beta-lactamase gene is present on the small plasmid RSF 1030 (R-Amp) . This same sequence is present on plasmid derivatives that have received a translocation of deoxyribonucleic acid specifying the TEM beta-lactamase and is also present on naturally occurring plasmids of the F1, F11, N, X, O, I, C, and W incompatibility groups that do not specify ampicillin resistance or specify O-type beta-lactamases.

Biochim Biophys Acta, 1975 Mar 28, 384(1), 242 - 9
Kinetic studies of a beta-lactamase by a computerized microacidimetric method; Labia R et al.; On-line computerized treatment of enzyme kinetic data allows the precise measurement of Michaelis--Menten constants (Km and V) from a single progress curve . This method has been used to determine the kinetic constants of a beta-lactamase extracted from an Escherichia coli strain . In the profile of enzymatic activity there obtained, Km and V are a function of the pH . From these results some information is derived about the mechanism of the enzyme--substrate binding.

Biochimie, 1975, 57(1), 29 - 34
{Purification of beta-lactamases by affinity chromatography}; Le Goffic F et al.; Affinity columns able to purifie beta-lactamases have been prepared either by linking covalently reversible inhibitors or substrates to agarose beds . The enzyme is eluted with a gradient of sodium chloride or released with the substrate . This method is a pertinent one for the purification of these enzymes and for the study of bacteria harbouring more than one beta-lactamase.

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