Beta lactamase

Jpn J Antibiot, 1983 Oct, 36(10), 2763 - 8
{Comparative study of the activities of beta lactamase inhibitors by nuclear magnetic resonance spectrometry}; O'hara K et al.; Nuclear magnetic resonance (NMR) spectrometry were applied for the measurement of inhibitory activity of some drugs towards beta-lactamase in living cells . From the integrated values of the signals based on 5-H and 6-H of beta-lactam ring or methyl proton of C-2 position of ABPC as substrate, the inhibition dose of 50% (ID50 value) to beta-lactamase activity under the condition containing 10 mg of ABPC was calculated . The ID50 values (mg) of clavulanic acid (CVA), N-formimidoylthienamycin (MK 0787) and dicloxacillin (MDIPC) known as beta-lactamase inhibitor were less than 0.0001, 0.4 and 0.6, respectively, when each inhibitor was preincubated for 10 minutes . The ID50 values of cefoxitin (CFX), cefmetazole (CMZ), latamoxef (LMOX) and cefotaxime (CTX) known as beta-lactamase resistant drugs were 1.4, 1.3, 0.4 and more than 20.0, respectively, under same condition . From these results, it was clearly shown that CVA is excellent inhibitor to the beta-lactamase of Richmond's class III in living cells . And the inhibitory effect of CFX, CMZ and LMOX was also clearly shown.

Med Res Rev, 1983 Oct-Dec, 3(4), 341 - 82
beta-Lactamase inhibitors; Cartwright SJ et al.; In summary, Table XVI shows the inhibition profiles of representative beta-lactamases from each major class of Richmond and Sykes . Either resistance (R) or sensitivity (S) is given as a general guide to the type of compounds likely to inhibit each class . Thus the (qualitative) statements regarding the effectiveness of clavulanic acid can be taken to represent those for the penam sulfones and similarly for MM4550 and the other olivanic acids, carpetimycins, PS series, and asparenomycins . This can also be said of cloxacillin and the other aromatic carboxamido penicillins . Compounds are also included which are specifically or particularly inhibitory to certain beta-lactamases.

J Antibiot (Tokyo), 1983 Oct, 36(10), 1372 - 9
Studies on 6-acetylmethylenepenicillanic acid (Ro 15-1903) . III . Relationship between in vitro activity and chemical stability; Arisawa M et al.; To resolve discrepancies between its enzymological activity and its in vitro or in vivo activity, 6-acetylmethylenepenicillanic acid (Ro 15-1903), a potent beta-lactamase inhibitor, was investigated for its chemical stability and its ability to penetrate the bacterial cell envelope . Although Ro 15-1903 was fairly stable in water or saline, it was found to be unstable in a rich medium, in mouse plasma and in human serum . Decomposition half-lives in Tryptic Soy Broth (TSB) and mouse plasma were determined by spectrometry to be 1.3 hours and 12 minutes respectively . These values were confirmed by a biochemical method for determination of Ro 15-1903 . Furthermore, a large enhancement of the in vitro activity was noticed when the assay medium was changed from TSB to a synthetic medium in which Ro 15-1903 was more stable . The ampicillin-potentiating activity marginally increased if a permeability mutant harboring the R6K plasmid, which codes for TEM-1 beta-lactamase production, was used instead of the wild-type strain . These results prove that the chemical instability of Ro 15-1903 is the main cause of its disproportionally low activity in vitro and in vivo . Ro 15-1903 is not nonspecifically inactivated by proteins, since it did not lose its activity after incubation with bovine serum albumin (50 mg/ml) for 2 hours at 37 degrees C . It seems to react specifically with beta-lactamase.

Presse Med, 1983 Sep 24, 12(33), 2035 - 7
{Branhamella catarrhalis in infectious pathology of the respiratory tract}; Le Faou A et al.; Branhamella catarrhalis has long been considered as a commensal of the upper respiratory tract . However, since 1972 several studies have shown that it can be responsible for respiratory tract infections . By isolating this micro-organism in sputum or nasal smear of 44 hospital patients we were able to confirm this finding, while delineating some of the clinical features of the infections observed . In all 44 patients these were mild infections, yet treatment appeared to be necessary in view of the risk of severe infection (B . catarrhalis septicaemia) in subjects with poor general condition . The germ is usually sensitive to antibiotics other than beta-lactam antibiotics (58% of the strains isolated were found to produce a beta-lactamase).

Gene, 1983 Sep, 24(1), 99 - 113
Escherichia coli RNA polymerase binding sites and transcription initiation sites in the transposon Tn3; Wishart WL et al.; We have identified the Escherichia coli RNA polymerase-binding sites and the transcription initiation sites in the transposon Tn3 . Results from nitrocellulose filter-binding assays indicate that there are two regions within Tn3 capable of forming stable binary complexes with RNA polymerase . The two regions are a 208-bp region containing the N-terminal coding sequence of the transposase (tnpA) and repressor (tnpR) genes, and a 332-bp region containing the N-terminal coding sequence for the beta-lactamase (bla) gene . DNase I footprint analysis of the 208-bp and 332-bp fragments further defined an extended region of protection, approx . 110 bp long, located between the transposase and repressor coding regions, and an 80-bp region of protection near the N-terminal coding sequence of the beta-lactamase gene . In vitro transcription studies with fragments containing these protected regions allowed us to determine the precise transcription initiation sites for the transposase, repressor, and beta-lactamase mRNAs . The transposase and repressor mRNAs are transcribed divergently and their transcription initiation sites are separated by 80 bp . The -35 homology regions for the transposase and repressor promoters are separated by 10 bp and the -10 homology region of the transposase promoter is coincident with the recombination site (res) for the site-specific recombinase activity (resolvase) of the repressor protein, which is required for resolution of Tn3 cointegrates . We discuss the significance of this complex divergently transcribed promoter region with respect to regulation of Tn3 transposition and we propose a model for coordinated regulation of the tnpA and tnpR genes . We also compare the Tn3 tnpA-tnpR intercistronic region with that of the closely related transposon gamma delta.

Aust N Z J Med, 1983 Aug, 13(4), 377 - 9
Empyema due to beta-lactamase-producing H . influenzae type b complicating severe laryngo-pharyngitis and cervical cellulitis; Allen RK et al.; A 70-year-old woman presented to hospital with an acute life-threatening cervical cellulitis and laryngopharyngitis followed by pneumonia . Despite a month's treatment with intravenous antibiotics including cephamandole she developed a bacteremic empyema due to a beta-lactamase producing H . influenzae . Subsequent treatment with chloramphenicol and surgical drainage was curative . The same pathogen was later isolated from her grandson's throat . Chloramphenicol is recommended as the drug of choice in such cases.

J Antimicrob Chemother, 1983 Aug, 12(2), 193 - 6
Isoelectric focusing of beta-lactamases on cellulose acetate membranes; Eley A et al.; Cellulose acetate membranes treated with boron trifluoride in methanol were found to provide suitable support material for isoelectric focusing of beta-lactamases; the method used is described . A linear pH gradient was obtained and the application of visible pI markers allowed the pH gradient to be monitored as focusing proceeded . beta-Lactamases extracted from antibiotic-resistant coliform organisms focused as sharp, straight bands upon visualization with nitrocefin . Results were known within two hours of applying the beta-lactamase extracts . The method allows a very economical use of expensive ampholyte and offers a quick and more simple alternative procedure for the identification of beta-lactamases than the conventional polyacrylamide gel method.

Antimicrob Agents Chemother, 1983 Aug, 24(2), 290 - 2
Penetration of sulbactam and ampicillin into peritoneal fluid; Wise R et al.; Twenty-five patients undergoing elective intraabdominal surgery received either 1 or 2 g of ampicillin together with 1 g of sulbactam intravenously before surgery . The peritoneal levels of the agent were measured . Both compounds penetrated peritoneal fluid readily; the mean percentage of penetration by ampicillin was 92%; that of sulbactam was 96% . After 1 g of each agent, the peritoneal levels of sulbactam were 47% greater than those of ampicillin . Our results suggest that 2 g of ampicillin plus 1 g of sulbactam should provide peritoneal levels that would inhibit most susceptible beta-lactamase-producing pathogens encountered in intraabdominal sepsis.

Proc Natl Acad Sci U S A, 1983 Aug, 80(16), 5080 - 4
Localization of a poliovirus type 1 neutralization epitope in viral capsid polypeptide VP1; van der Werf S et al.; Poliovirus type 1 cDNA sequences coding for viral capsid polypeptide VP1 were inserted into the beta-lactamase sequence of Escherichia coli plasmid pBR322 . Resulting recombinant plasmid pSW119 expressed in Escherichia coli a VP1-beta-lactamase fusion protein that reacted with antibodies raised against poliovirus capsid polypeptide VP1 and with a monoclonal poliovirus type 1 neutralizing antibody, C3 . Deletions of various lengths were generated within the VP1 sequence . The hybrid proteins expressed by the deleted plasmids did not react any more with C3 when the region of VP1 amino acids 95-110 (poliovirus nucleotides 2,754-2,806) was deleted . Therefore, the C3 epitope responsible for virus neutralization is most probably located in this region of the capsid polypeptide.

Arch Otolaryngol, 1983 Aug, 109(8), 533 - 5
Treatment of ampicillin-resistant acute otitis media in the chinchilla; Reilly JS et al.; The efficacy of sulbactam sodium (CP45,899-2) was investigated using the chinchilla animal model of acute otitis media with effusion (AOME) . Both ears of 78 chinchillas were inoculated with beta-lactamase-producing nontypable Hemophilus influenzae . Half of the animals were treated with ampicillin sodium alone (group A) and the remaining animals received ampicillin plus sulbactam (group B) . On day 14, all of the ears in group B were culture-negative whereas H influenzae was recoverable in over 70% of the effusions in group A . Similarly, the course of middle ear effusion was significantly abbreviated in group B during the two-week study period . These findings suggest that sulbactam in combination with ampicillin is effective in treating AOME secondary to infection with beta-lactamase-producing nontypable H influenzae in the chinchilla animal model.

Braz J Med Biol Res, 1983 Jul, 16(2), 81 - 7
Purification and properties of a new beta-lactamase from Streptomyces UCSM-104; Garces E et al.; Extracellular beta-lactamase was produced in larger amounts by Streptomyces UCSM-104 than by Streptomyces albus G or strains R-39 and K-11 . The anionic character displayed by the enzyme from Streptomyces USCM-104 on DEAE-cellulose chromatography was consistent with its mobility in polyacrylamide gel electrophoresis . This beta-lactamase is a low molecular weight (14,800) globular protein having approximately 124 amino acid residues . The enzyme behaved as a penicillinase toward several substrates studied, being most active on benzylpenicillin and ampicillin . On the other hand, it was inhibited by methicillin and cloxacillin, which behaved as competitive inhibitors for the hydrolysis of benzylpenicillin and cephaloridine.

Gene, 1983 Jul, 23(1), 85 - 93
Expression of poliovirus capsid polypeptide VP1 in Escherichia coli; van der Werf S et al.; A recombinant plasmid (pPV1-958) carrying a poliovirus cDNA insert corresponding to nucleotides 1-5750 of the poliovirus RNA genome was constructed through in vitro recombination of two plasmids carrying overlapping poliovirus cDNA inserts (Van der Werf et al., Proc . Natl . Acad . Sci . USA 78 (1981) 5983-5987) . pPV1-958 was then cleaved with PstI and the fragment carrying the sequence encoding capsid polypeptide VP1 was isolated and subcloned at the PstI site of pBR322 . The VP1 sequence was successfully fused with the 5' sequence of the beta-lactamase gene by deleting 300-350 bp from each side of the PvuI site of the plasmid with nuclease BAL31 . One of the resulting plasmids, pSW119, expressed an Mr 49 000 VP1-beta-lactamase-fusion protein, which was specifically immunoprecipitated with anti-VP1 immune serum but not by hyperimmune sera raised against native or heat-inactivated virus.

Sem Hop, 1983 Jun 30, 59(26), 1959 - 62
{Moxalactam: an inactivator of beta-lactamases}; Labia R; At present, Moxalactam is the beta-lactam antibiotic which shows the greatest resistance to beta-lactamases . Moxalactam has little, if any, interactions with penicillinases, as is also the case of cefoxitin, cefuroxime and cefotaxime . Resistance of Moxalactam to cephalosporinases, however, appears to be related to the unique property of inactivating them . This implies: a specific process which involves the active site of the enzymes; a progressive, or time-dependent, process, which progressively abolishes beta-lactamase activity . This process was observed with most of the tested cephalosporinases . Thus, Moxalactam is the first beta-lactam antibiotic which inactivates beta-lactamases . This is probably why Moxalactam is so resistant to the action of beta-lactamases.

Science, 1983 Jun 17, 220(4603), 1285 - 8
Localization of a Plasmodium surface antigen epitope by Tn5 mutagenesis mapping of a recombinant cDNA clone; Lupski JR et al.; A recombinant complementary DNA clone from Plasmodium knowlesi makes a beta-lactamase fusion polypeptide in Escherichia coli that reacts with a monoclonal antibody to a Plasmodium surface antigen . An epitope of the surface antigen was localized by transposon Tn5 mutagenesis mapping of the complementary DNA clone . The Tn5 mutation having the farthest 5' insert into the complementary DNA portion of the chimeric gene, giving the shortest truncated protein that maintained the ability to bind monoclonal antibody, defined the location of the epitope.

J Antimicrob Chemother, 1983 Jun, 11(6), 583 - 7
The elimination of sulbactam alone and combined with ampicillin in patients with renal dysfunction; Wright N et al.; Sulbactam, a beta-lactamase inhibitor, was given as a single agent to 12 subjects with varying degrees of renal dysfunction . The half-life in normal subjects was 1.1 h and increased to 21.3 h in those with terminal renal failure . Sulbactam together with ampicillin was given to a further five subjects . It was concluded that if the two agents are given together, the plasma ratio of one to the other will remain constant whatever the renal function.

Infection, 1983 May-Jun, 11(3), 150 - 4
Pharmacokinetics of amoxicillin and flucloxacillin following the simultaneous intravenous administration of 4 g and 1 g, respectively; Adam D et al.; The combination of amoxicillin and flucloxacillin not only widens the spectrum of pathogenic organisms covered by either of the substances alone; synergy has also been observed, particularly against beta-lactamase-producing organisms . For this reason, the possible interaction of these two penicillins regarding their pharmacokinetics was investigated with respect to therapeutic application . The parameters were calculated on the basis of an open two-compartment model . The highest serum levels of amoxicillin from 551 to 1074 mg/l when 4 g were administered alone, and from 403 to 1133 mg/l when administered together with 1 g flucloxacillin . Flucloxacillin concentrations ranged from 118 to 357 mg/l when administered alone, and from 151 to 226 mg/l in the presence of amoxicillin . Thus, there is no significant difference in the peak levels of either substance when given alone or in combination . The pharmacokinetic parameters of both substances basically do not depend on the presence of the other . A slight decrease was observed in the distribution rate of amoxicillin from the central to the peripheral compartment in the presence of flucloxacillin . Its relevance is questionable, however, since the effect was only minor.

Biochem J, 1983 May 1, 211(2), 447 - 54
Mechanism of inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid; Arisawa M et al.; The interaction between 6-acetylmethylenepenicillanic acid (compound Ro 15-1903; AMPA) and TEM-1 beta-lactamase was investigated in order to elucidate the mechanism of action of AMPA . Formation of the enzyme-inhibitor complex (EA) was accompanied by a shift of the absorbance maximum from 292 nm to 303 nm and an increase in the absorption . Regeneration of activity was very slow and incomplete, reaching about one-third of the initial activity after 48 h at 37 degrees C . This behaviour indicated a branched pathway of the decay of the inactivated enzyme . Kinetic and isoelectric-focusing experiments proved this assumption . The first-order constant of regeneration of active enzyme was 6 X 10(-6)-10 X 10(-6) s-1, whereas the rate constant leading to inactive enzyme (EA') was 10 X 10(-6)-15 X 10(-6) s-1 at pH 7.0 . Both constants became larger at higher pH . Inactive enzyme (EA') consisted of two major species, with pI 5.36 (EA'1) and 5.30 (EA'2) . The former increased at the beginning of incubation but decreased after prolonged incubation . From consideration of these results and previous data {Arisawa & Then (1983) Biochem . J . 209, 609-615}, a likely mechanism of inactivation of TEM-1 beta-lactamase by AMPA is discussed.

Nature, 1983 Apr 7, 302(5908), 536 - 8
Cloning and expression in E . coli of the malarial sporozoite surface antigen gene from Plasmodium knowlesi; Ellis J et al.; The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s) . Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man . Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species . One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s) . However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained . To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library . From this library we have isolated a clone that expresses the sporozoite surface antigen as a beta-lactamase fusion protein in the plasmid pBR322 . This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.

J Pharm Sci, 1983 Apr, 72(4), 331 - 5
Further studies on the catalysis of hydrolysis and aminolysis of benzylpenicillin by metal chelates; Tomida H et al.; It was suggested previously that the very rapid catalysis of benzylpenicillin hydrolysis and aminolysis by zinc ion and tris(hydroxymethyl)aminomethane (tromethamine) was mediated by a ternary complex in which the metal ion not only held the substrate and tromethamine in close proximity but also lowered the pKa of a bound tromethamine hydroxyl group making it a very powerful nucleophile . In this study the scope of this reaction was explored further by examining the effects of changes in substrate side chain, metal ion, and amino alcohols . All of the penicillins studied showed about the same rate of reaction . Of the other metal ions examined Cu2+ and Ni2+ showed no activity, Mn2+ very slight activity, and Cd2+ and Co2+ somewhat greater activity . The latter was the most effective of this group but was 40 times slower than zinc . The results with a number of amino alcohols provided additional evidence for the ternary complex mechanism . Studies with the methyl ester of benzylpenicillin indicated that the metal ion is bound to the antibiotic at the carboxylate site and that a different mechanism is involved in the slower catalysis observed with the ester . Some comparison is made with a zinc-dependent beta-lactamase.

Antimicrob Agents Chemother, 1983 Apr, 23(4), 622 - 5
Selection and characterization of beta-lactam-resistant Escherichia coli K-12 mutants; Jaffe A et al.; beta-Lactam-resistant mutants of Escherichia coli K-12 were selected by using 12 different beta-lactam derivatives . The mutants fell into three categories showing (i) altered permeation through reduction or loss of outer membrane porin proteins (including ompF, ompR, and envZ alleles); (ii) increase in the rate of synthesis of chromosomally mediated beta-lactamase; or (iii) defective synthesis or action of cyclic adenosine 3',5'-phosphate (cya and crp alleles).

Postgrad Med, 1983 Mar, 73(3), 187 - 91
Hemophilus influenzae respiratory infection in adults . 2 . Treatment guidelines; Parker RH; Once a Hemophilus influenzae isolate is identified as the cause of a respiratory tract infection in an adult, it should be tested for beta-lactamase production, ie, for ampicillin resistance . The incidence of ampicillin-resistant strains of H influenzae is increasing . The Centers for Disease Control in Atlanta estimates an average incidence nationwide of 18% to 22%; the rate varies considerably from community to community . Thus, practitioners should be aware of the ampicillin-resistance rate in their community and should keep this rate in mind especially when treating patients empirically . Patients with H influenzae infections who are acutely ill, who fail to respond to ampicillin, or who are known to have an ampicillin-resistant infection on the basis of laboratory findings should receive therapy designed to combat ampicillin-resistant strains.

Arch Biochem Biophys, 1983 Mar, 221(2), 585 - 9
Synthesis of a human leukocyte interferon with a modified carboxy terminus in Escherichia coli; Chang NT et al.; A recombinant plasmid was constructed that results in deletion of eleven COOH-terminal amino acids of human leukocyte A interferon and their replacement by nine unrelated amino acids encoded by the beta-lactamase gene of pBR322 . This altered human leukocyte interferon exhibits antiviral activity slightly lower than the natural molecule and appears to be more susceptible to proteolytic degradation as well . These results also confirm the conclusion that the eleven COOH-terminal amino acids are not essential for antiviral or antiproliferative activity.

Biochem J, 1983 Mar 1, 209(3), 609 - 15
Inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid; Arisawa M et al.; 6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases . Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated . 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1 . The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin . Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15 . Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme . All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.

Antimicrob Agents Chemother, 1983 Mar, 23(3), 407 - 12
Tn2011, a new transposon encoding oxacillin-hydrolyzing beta-lactamase; Nakazawa H et al.; The type II penicillinase (oxacillin-hydrolyzing beta-lactamase, OXA-1) gene on plasmid Rms213 was transposed to various plasmids or to the host chromosome . The transposon bearing this gene, designated Tn2011, conferred resistance to ampicillin, streptomycin, sulfonamide, and mercuric chloride . By restriction endonuclease digestion and agarose gel electrophoresis, the molecular weight of Tn2011 was estimated to be 12.5 X 10(6) . The transposition frequency of Tn2011 was about 10(-4) to 10(-5) . The activity of type II penicillinase is related to the copy number of the replicon bearing Tn2011.

Cell, 1983 Mar, 32(3), 809 - 16
Insertion of IS2 creates a novel ampC promoter in Escherichia coli; Jaurin B et al.; A class of ampC beta-lactamase-hyperproducing mutants of Escherichia coli were shown to have the insertion element IS2 inserted into the ampC promoter . The insertion of IS2 in orientation II created a novel promoter in which the -35 region and the 17 bp long spacing sequence between the two consensus sequences are present in IS2 DNA, whereas the -10 region from the original ampC promoter is retained . In vitro transcription revealed that the transcription initiation site in the ampC::IS2 mutants was identical with that of ampC wild-type promoter . The novel promoter exhibited a 20-fold increase in promoter strength relative to the original ampC promoter, presumably due to the increase in the spacing sequence from 16 to 17 bp . The evolution of transposable elements and of control elements such as promoters are discussed on the basis of the findings described herein.

Acta Pathol Microbiol Immunol Scand {B}, 1983 Feb, 91(1), 49 - 54
Resistance types in Escherichia coli . III . beta-lactamase production; Sogaard P; The beta-lactamase activity with ampicillin (A) and cephalothin (Ce) as substrate was examined in 22 E . coli strains by a micro-iodometric - (MIA) and an ultraviolet assay (UVA) . The strains were divided into three groups according to their sensitivity to A and carbenicillin (Ca) . Group 1 contained 6 A-Ca sensitive (A-s/Ca-s) strains, group 2 contained 8 A-resistant (A-r)/Ca-s strains and group 3 eight A-r/Ca-r ones . MIA with A as substrate showed high activity in group 3 . The activities in groups 1 and 2 did not differ and were very low . Group 1 had the lowest and group 3 the highest IC50 with A . UVA with Ce as substrate showed high activity in group 2, lower in group 3 and lowest in group 1 . IC50 corresponded to this order . MIA with Ce as substrate showed high activity (measured 25 min after enzyme was added) in group 3, lower activity in group 2 and lowest in group 1 . The initial velocity was highest in group 2, but decreased with time . In group 3 the velocity increased with time . Group 2 (A-r/Ca-s) strains contained cephalothinases that apparently were inhibited by iodine . The beta-lactam resistance was probably chromosomally mediated . Group 3 (A-r/Ca-r) strains contained beta-lactamases with predominantly ampicillinase activity and were not inhibited by iodine . The A-resistance was probably plasmid mediated.

C R Seances Acad Sci III, 1983 Jan 17, 296(2), 119 - 22
{Beta-lactamase synthesis and excretion in a non-leaky wild strain and a leaky mutant of Escherichia coli K-12}; Fognini-Lefebvre N et al.; After transformation of Escherichia coli strains with plasmid pBR 322 and growth in rich L medium, the total amount of beta-lactamase produced, strongly decreased when the temperature was raised from 30 to 42 degrees C, but increased after addition of ampicillin or tetracycline to the medium . beta-lactamase was synthesized and exported into the periplasmic space of wild-type strain, but was not significantly released into the extracellular medium, after growth at low temperature . We have identified an E . coli mutant which excreted up to 90% of total amount of beta-lactamase activity, any temperature . This mutant has been used as an indicator strain, for the development of an in situ test allowing the detection of beta-lactamase excretion.

Infection, 1983, 11 Suppl 2, S71 - 3
The influence of beta-lactamases on minimal inhibitory concentrations; Wiedemann B et al.; The stability towards hydrolysis by beta-lactamases is commonly used as a major criterion for the quality of a beta-lactam antibiotic . The activity of a beta-lactam compound is influenced by many factors, however . After the introduction of third generation cephalosporins, evidence arose that beta-lactamases can be the only reason for resistance, although the drug is not or only slightly hydrolyzed by the corresponding enzyme . Here it seems as if binding of the antibiotic to the beta-lactamase is the main factor which influences the drug's activity . But, in fact, the situation is much more complicated, as at least several different factors must be considered in combination with beta-lactamase activity.

Scand J Infect Dis, 1983, 15(2), 225 - 6
Transfer of beta-lactamase production in Branhamella catarrhalis; Kamme C et al.; A beta-lactamase positive Branhamella catarrhalis strain, B.c . 002 PcR, is described . The strain has the ability to transfer beta-lactamase production . The strain was isolated from nasopharynx in an adult patient with long-standing laryngitis . It showed atypical colony morphology.

Ann Microbiol (Paris), 1983 Jan-Feb, 134A(1), 29 - 38
{Affinity of N-formimidoyl-thienamycin (MK-0787) for beta-lactamases, analysis by a statistical method: correspondence analysis}; Labia R et al.; N-formimidoyl-thienamycin (MK-0787) is not subject to beta-lactamase hydrolysis . Nevertheless, this compound does show some affinity for the TEM-type penicillinases, cephalosporinases and a few other beta-lactamases . Surprisingly no affinity was found for the penicillinases of the CARB-type . The results thus obtained for affinity constants Ki and Km were submitted to a statistical analysis treatment, i.e . correspondence analysis and hierarchic ascendent classification . This confirms the fact that MK-0787 presents an unusual behaviour towards beta-lactamases.

J Cell Biochem, 1983, 22(3), 141 - 9
Specific processing of the bacterial beta-lactamase precursor in Saccharomyces cerevisiae; Roggenkamp R et al.; Synthesis and processing of the bacterial enzyme beta-lactamase (E.C . 3.5 . 2.6) were studied in Saccharomyces cerevisiae . The 2-micron DNA vector pADH040-2 containing the yeast ADH1 promoter fused to the bacterial gene was used in order to obtain enhanced synthesis of the bacterial protein in yeast transformants . Both precursor and mature beta-lactamase were shown to be present in yeast cells, the precursor being the major product . The mature enzyme was purified about 500-fold over crude extracts to apparent homogeneity and thus represents nearly 0.2% of the total yeast protein . No difference in specific activity and molecular weight could be observed when compared with the authentic beta-lactamase from Escherichia coli . Specificity of the processing of beta-lactamase in yeast cells was verified by partial amino acid sequence analysis demonstrating the removal of the signal peptide at the correct position.

Scand J Infect Dis, 1983, 15(4), 407 - 8
Plasmid-mediated beta-lactam resistance of Escherichia coli isolated from travellers' diarrhoea in the Far East; Moller JK et al.; The transfer of drug resistance and beta-lactamase activity of 28 E . coli from travellers' diarrhoea were examined . Plasmid-mediated beta-lactam resistance was found in 18 and at least 3 different types of beta-lactamases were demonstrated.

Z Allg Mikrobiol, 1983, 23(6), 367 - 74
{Replication and expression of plasmid pBR 322 during discontinuous culture of a stringent and relaxed Escherichia coli strain}; Hecker M et al.; The E . coli strains CP78 and CP79 carrying the plasmid pBR 322 display similar growth kinetics in discontinuous culture . During limitation of amino acids the stringent strain CP78 is able to synthesize guanosine-5'diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3' diphosphate, but the relaxed strain can not produce highly phosphorylated guanosine nucleotides . During the logarithmic phase of growth both strains contain similar amounts of plasmid DNA . During amino acid starvation plasmid DNA is amplified in the relaxed strain only, whereas in the stringent strain the plasmid content per cell remains constant . In stationary phase cells of CP78 a higher activity of plasmid-coded beta-lactamase than in CP79 cells was detected . Furthermore, remarkable differences between both strains were observed in the composition of proteins derived from the periplasmic fraction and separated by polyacrylamide gel electrophoresis . Our results might indicate a negative control of pBR 322 DNA replication by ppGpp during amino acid starvation.

DNA, 1983, 2(1), 23 - 30
Differential expression of cloned mouse metallothionein sequences in Escherichia coli; Mbikay M et al.; A cDNA for mouse hepatic metallothionein I has been cloned into pBR322 (Mbikay et al., 1981) . Although this recombinant plasmid (M135) possesses the metallothionein sequence in the same reading frame as that of beta-lactamase, it fails to direct the synthesis of a fused beta-lactamase-metallothionein protein in Escherichia coli . Another plasmid (M244) was derived from M135 by deleting an internal 390-bp segment made of the 5' noncoding region of metallothionein, the dG-dC tail, and some beta-lactamase sequences . Bacteria harboring the new plasmid now contain in their periplasmic space a cysteine-rich, cadmium-binding protein of 12,000 daltons, as expected . These observations demonstrate that expression of cloned DNA in bacteria could depend as much on its primary structure as on proper insertion in relation to a promoter.

J Bacteriol, 1983 Jan, 153(1), 27 - 32
Role of primary structure and disulfide bond formation in beta-lactamase secretion; Pollitt S et al.; Plasmid pBR322-encoded beta-lactamase was shown to contain a single disulfide bond, which caused the protein to migrate faster in sodium dodecyl sulfate-polyacrylamide gels than the fully reduced form . A similar difference in mobility of the in vitro synthesized precursor before and after reduction indicates that it also contained a disulfide bond . Formation of the disulfide bond in vivo, however, occurred concomitant with processing . In vivo accumulation of the precursor by inhibition of secretion did not allow disulfide bond formation to occur . This result is consistent with post-translational translocation of the precursor . Synthesis of a fragment of beta-lactamase lacking the carboxy terminus was obtained by insertion of a foreign DNA segment into the PstI site of bla . Processing and secretion of the protein did not appear to be greatly affected, indicating that the carboxy terminus is not required for secretion.

DNA, 1983, 2(4), 255 - 64
Cloning and expression in Escherichia coli of full-length complementary DNA coding for human alpha 1-antitrypsin; Bollen A et al.; A cDNA library prepared from human liver was screened for alpha 1-antitrypsin, a major constituent of plasma which functions as inhibitor of proteolytic enzymes . The library was screened using a 12-base-long synthetic oligodeoxyribonucleotide corresponding to a known DNA fragment of human alpha 1-antitrypsin and by hybrid-selection of alpha 1-antitrypsin mRNA . A plasmid, pULB1523, was identified carrying a cDNA insert of about 1400 bp coding for human alpha 1-antitrypsin . Restriction mapping and DNA sequence analysis indicated that the 1400 bp code for the signal peptide and for the complete mature alpha 1-antitrypsin molecule . In addition, a solid-phase enzyme-linked immunoassay showed that pULB1523 expresses human alpha 1-antitrypsin in bacteria . Fusion of the alpha 1-antitrypsin sequence to the leader sequence of the beta-lactamase gene (plasmid pKT287) resulted also in the expression of the protein in bacteria.

Mol Gen Genet, 1983, 189(2), 282 - 8
Tn2610, a transposon involved in the spread of the carbenicillin-hydrolyzing beta-lactamase gene; Yamamoto T et al.; We have found a new transposon, Tn2610, on pCS200 in clinical isolates of Escherichia coli, which encodes the carbenicillin-hydrolyzing beta-lactamase gene in combination with the resistance determinants to streptomycin and sulfonamide . Tn2610 has a molecular size of 24 kilobase pairs and is flanked by long inverted repeat sequences of 3 kilobase pairs in length . Genetical and physical analyses indicate that Tn2610 is a single transposable unit encoding the multiple resistance determinants and that is different from any previously described transposon . The characteristic DNA structure observed in various complex resistance transposons involved in the transposition of the carbenicillin-hydrolyzing beta-lactamase gene is discussed.

An Acad Bras Cienc, 1982 Dec, 54(4), 629 - 34
Kinetic study on beta-lactamase from Streptomyces UCSM-104; Garces E et al.; The beta-lactamase of Streptomyces UCSM-104 behaves as a penicillinase with the different substrates studied, being more active with benzylpenicillin (KM 2.6 mM) and ampicillin (KM 1.5 mM) . On the other hand, it was competitively inhibited by methicillin (Ki 0.035 mM) and cloxacillin (Ki 0.35 mM) using benzylpenicillin as substrate . According to the criterion of Jack and Richmond in relation to the degree of inhibition obtained, the enzyme was found to be resistant to cloxacillin and sensible to methicillin.

Biochem J, 1982 Dec 1, 207(3), 429 - 36
Interaction of clavulanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39; Frere JM et al.; The reactions of beta-lactamases of Actinomadura R39 and Streptomyces albus G with clavulanate proceed along branched pathways . Both enzymes perform the hydrolysis of this beta-lactam with rather high efficiencies (kcat . = 18s-1 and 52s-1 respectively) . If large clavulanate/enzyme ratios are used, complete inactivation of the enzymes is observed . At lower ratios, inactivation is only partial . Irreversible inactivation occurs after 400 and 20000 turnovers for the A . R39 and S . albus G enzymes respectively . With the A . R39 beta-lactamase, a transiently inhibited complex is also formed that remains undetectable with the S . albus G beta-lactamase . Kinetic models are presented and studied for the interaction between clavulanate and both enzymes . A tentative general reaction scheme is also discussed.

Nucleic Acids Res, 1982 Nov 11, 10(21), 6715 - 32
The construction of cosmid libraries which can be used to transform eukaryotic cells; Grosveld FG et al.; Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E . coli guanine phosphoribosyltransferase gene) . The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids . Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method . Several clones from the human beta-globin locus were isolated . These cosmids transform mouse L cells at high efficiency in both circular and linear form . The newly introduced genes are expressed accurately in L cells.

Am Fam Physician, 1982 Nov, 26(5), 153 - 7
Hemophilus influenzae bronchitis and pneumonia; Gleckman R et al.; Hemophilus influenzae is increasingly recognized as an important pathogen in a wide spectrum of diseases in adults . It is often implicated in the infectious exacerbation of chronic bronchitis, and it is a frequent cause of pneumonia in adults . The recent recognition of ampicillin-resistant strains should be a reminder to obtain beta-lactamase or antibiotic susceptibility testing when H . influenzae is isolated in specimens from patients with lower respiratory infections.

Proc Natl Acad Sci U S A, 1982 Nov, 79(21), 6409 - 13
Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function; Dalbadie-McFarland G et al.; We have used oligonucleotide-directed mutagenesis to make a specific change in the beta-lactamase (EC 3.5.2.6) (ampicillin resistance) gene of the plasmid pBR322 . Evidence suggests that the active site for this enzyme may include a serine-threonine dyad (residues 70 and 71) . By priming in vitro DNA synthesis with a chemically synthesized 16-base oligodeoxyribonucleotide, we have inverted the Ser-Thr dyad to Thr-Ser and thereby generated a mutant with an ampicillin-sensitive phenotype . This "double-mismatch" method is relatively simple and also very general because detection of mutants is at the level of DNA and involves only colony hybridization . Accordingly, the procedure can be applied to any DNA sequence and does not depend on the phenotype of the mutant.

Gene, 1982 Nov, 20(1), 1 - 10
Transcription of the bacterial beta-lactamase gene in Saccharomyces cerevisiae; Breunig KD et al.; We have examined the transcription in yeast of Escherichia coli-yeast 2-micrometers DNA recombinant plasmids carrying the bacterial beta-lactamase (bla) gene . In Saccharomyces cerevisiae both strands of the gene are transcribed giving multiple RNA species of distinct lengths . At least one RNA transcript derived from the coding strand initiates at a yeast promoter on the 2-micrometers DNA segment . Another mRNA of 1.1 kb starts right in front of the gene on the bacterial DNA sequence . Deletion experiments have shown that expression of the bacterial bla gene is dependent on the presence of bacterial sequences right in front of the gene . Mutants lacking the bacterial promoter region do not give detectable gene products in yeast . The expression can be restored by substituting for the deleted sequence a DNA fragment which carries the E . coli lac promoter-operator region . We conclude that the bacterial promoter region of the bla gene as well as the lac promoter-operator fragment have promoter activity in yeast and that yeast-bla fusion transcripts cannot be used as a functional messenger for beta-lactamase in yeast.

J Antibiot (Tokyo), 1982 Nov, 35(11), 1578 - 83
6-Acetylmethylenepenicillanic acid (Ro 15-1903), a potent beta-lactamase inhibitor . I . Inhibition of chromosomally and R-factor-mediated beta-lactamases; Arisawa M et al.; 6-Acetylmethylenepenicillanic acid (Ro 15-1903) was seen to be a powerful inhibitor of various beta-lactamases . Nearly all of the chromosomally and R-factor-mediated beta-lactamases which were studied were inhibited at much lower concentrations than required with clavulanic acid or sulbactam . Ro 15-1903 protected beta-lactamase-labile penicillins and cephalosporins from hydrolysis . The compound itself was stable against hydrolysis and inhibition of beta-lactamase was irreversible.

J Biol Chem, 1982 Oct 25, 257(20), 11860 - 3
A bacterial secretory protein requires signal recognition particle for translocation across mammalian endoplasmic reticulum; Muller M et al.; In vitro transcription of DNA from plasmid pBR322 was coupled to cell-free translation in a wheat germ system . The major translation product was pre-beta-lactamase . Upon addition of dog pancreas microsomes, the precursor was processed to authentic beta-lactamase as shown by partial NH2-terminal sequence analysis . Processing was linked to translocation into the microsomal vesicles . Salt-extracted microsomes did not process pre-beta lactamase but could be reactivated by purified signal recognition particle, which is the functional component of the salt wash (Walter, P., and Blobel, G . (1980) Proc . Natl . Acad . Sci . U . S . A . 77, 7112-7116) . Signal recognition particle alone caused a drastic translation arrest that could be released by salt-depleted membranes . These data are consistent with those obtained for eukaryotic proteins and suggest that co-translational translocation of both bacterial and eukaryotic secretory proteins across the endoplasmic reticulum require identical components.

Biochim Biophys Acta, 1982 Oct 11, 721(2), 185 - 90
Liposome-mediated DNA transfer in eukaryotic cells . Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage; Nicolau C et al.; The pBR322 plasmid containing the sequence encoding beta-lactamase, the enzyme conferring resistance to ampicillin, was encapsulated in liposomes of different phospholipid composition and incubated with synchronized cells . In mitotic cells as compared to cells synchronized in G1, twice as many exogeneous DNA molecules were found associated with the cell nuclear DNA, when fluid, neutral liposomes were used . These liposomes are taken up by the cells mainly via endocytosis . When fluid, negatively charged liposomes were used as carriers about the same number of exogenous DNA molecules were found associated with the nuclear DNA both in mitotic and in G1-synchronized cells . The efficiency for gene transfer of liposomes entering the cells by different mechanisms was further studied and expressed both by the fraction of the radioactive plasmid associated with the nuclear DNA and by the level of the beta-lactamase activity detected in the transfected cells . It appears that liposomes entering the cells mainly via an energy-dependent mechanisms are more efficient for this type of DNA transfer.

Br J Vener Dis, 1982 Oct, 58(5), 317 - 20
Quinolone derivative, flumequine, as short-term treatment for gonorrhoea; Svindland HB et al.; The antigonococcal activity of the quinolone derivative flumequine was evaluated . Of 246 strains examined, 240 (97.5%) strains showed minimum inhibitory concentrations (MICs) of flumequine of less than or equal to 0.4 microgram/ml, including three beta-lactamase-producing strains . The six remaining strains showed MICs from 3.2 to 9.6 micrograms/ml . By disc diffusion tests using 3-micrograms discs of flumequine the zones of growth inhibition correlated well with the MICs of flumequine . The effect of treatment with flumequine was compared in 239 patients with uncomplicated gonorrhoea . A single dose regimen of 1200 mg flumequine orally, a two-dose regimen of 1200 and 800 mg, and a three-dose regimen of 1200, 800, and 800 mg (six hours apart) were given . With a single dose of flumequine the failure rate was 26% . The two-dose and three-dose regimens were equally effective with an overall cure rate of 95.4% . In patients harbouring beta-lactamase-producing gonococci the infection was cured . The failures (10 men) included all of the six patients infected with flumequine-resistant gonococci . Side effects were noted by 14.6% of the patients and were mostly described as dizziness.

Mol Cell Biol, 1982 Sep, 2(9), 1044 - 51
Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells; Gorman CM et al.; We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells . The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted . Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells . Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells . To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed . Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type . We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

J Biol Chem, 1982 Aug 10, 257(15), 9205 - 10
Precise location of two promoters for the beta-lactamase gene of pBR322 . S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro; Brosius J et al.; We identified two promoters for the beta-lactamase gene of plasmid pBR322 . RNA isolated from bacteria containing pBR322 or RNA transcribed in vitro on pBR322 templates was hybridized to 5' end-labeled single-stranded plasmid probes (Berk, A . J., and Sharp, P . A . (1977) Cell 12, 721-732) . Electrophoretic analysis of the nuclease S1 digestion products next to Maxam-Gilbert sequencing ladders closely defines the transcriptional initiation points . The natural promoter lies near the coding sequence of the beta-lactamase gene, initiating transcription at -35 bases before the ATG initiation codon, while a second promoter initiates at positions -244 and/or -245 (on the opposite side of the Eco RI site) . This promoter overlaps the promoter transcribing in the opposite direction toward the tetracycline gene(s) and starts in the -10 region of that promoter . S1 mapping of procaryotic mRNA, transcribed in vivo, allows both an accurate identification of promoters and the analysis of their transcriptional regulation.

Biochem J, 1982 Aug 1, 205(2), 413 - 7
The interaction of anthraquinone dyes with the plasmid-mediated OXA-2 beta-lactamase; Monaghan C et al.; Although beta-lactamases do not require any nucleotide co-substrates, the OXA-2 type is inhibited competitively by Cibacron Blue 3GA, and by other anthraquinone dyes, including some simpler compounds with no side chain . The enzyme causes a red shift in the spectrum of Cibacron Blue . The beta-lactamase can be adsorbed in Blue Sepharose and specifically eluted by benzylpenicillin . These results indicate that the binding of anthraquinone dyes is a specific effect similar to that seen with many nucleotide-binding enzymes.

Antimicrob Agents Chemother, 1982 Aug, 22(2), 208 - 12
Effects of ampicillin plus sulbactam on bowel flora in patients undergoing colorectal surgery; Kager L et al.; The influence of a combination of ampicillin and a beta-lactamase inhibitor, sulbactam, on colonic bacterial flora was studied in patients undergoing colorectal surgery . Drug concentrations were determined in serum, intestinal mucosa, and feces . The results of the drug concentrations were correlated with changes in the numbers of aerobic and anaerobic bacterial bowel flora . Ampicillin and sulbactam were given intravenously to 21 patients in 500-mg doses of each drug every 8 h for 2 days . The first dose was given when anesthesia was begun . The concentrations of ampicillin in serum were significantly higher (0.01 greater than P greater than 0.001) than those of sulbactam . In contrast, at all time intervals, the concentrations of sulbactam in intestinal mucosa were significantly higher than those of ampicillin (0.05 greater than P greater than 0.01) . The serum half-life of the two drugs was the same at 2.2 h . After one dose of the antibiotics, sulbactam was detectable in the feces of four patients, and ampicillin was detectable in the feces of 11 patients . During the 2 days of ampicillin and sulbactam prophylaxis, no measurable decrease occurred in the anaerobic flora of the bowel; a mean decrease of 3 logs occurred in the anaerobic bowel flora . The results of this study show that ampicillin and sulbactam prophylaxis had a greater effect on the anaerobic bowel flora than on the aerobic bowel flora . In light of the in vitro spectrum of ampicillin and sulbactam activity, it was surprising that bowel aerobic flora was unchanged.

J Biol Chem, 1982 Jul 25, 257(14), 7930 - 2
Targeted point mutation that creates a unique Eco RI site within the signal codons of the beta-lactamase gene without altering enzyme secretion or processing; Charles AD et al.; A method has been developed for constructing site-specific mutations by using a strongly selectable marker on which to "piggy-back" a desired mutation that may be phenotypically silent . Using this approach, a new unique Eco RI restriction site has been generated at the beginning of the signal codons of the beta-lactamase gene of the plasmid pBR322 . The consequential alteration of the second amino acid of the signal from Ser to Arg has no effect on either the transport or the processing of the beta-lactamase.

Biochemistry, 1982 Jun 8, 21(12), 2857 - 62
Inhibition of the RTEM beta-lactamase from Escherichia coli . Interaction of the enzyme with derivatives of olivanic acid; Easton CJ et al.; From chemical and kinetic studies of the interaction of the RTEM beta-lactamase from Escherichia coli with three derivatives of olivanic acid, MM22382 (1), MM13902 (2), and MM4550 (3), a mechanism for the inhibition of the enzyme by these compounds is proposed: the interaction proceeds by formation of an acyl-enzyme, the delta 2-pyrroline, which may either deacylate or undergo tautomerization to the more tightly bound delta 1-pyrroline . The ability of olivanic acids to inhibit the enzyme thus depends on the partitioning of the acyl-enzyme to the delta 1-pyrroline ( a process that competes with the normal hydrolytic pathway) and on the rate of regeneration of free enzyme from this complex.

Eur J Biochem, 1982 Jun, 124(3), 561 - 6
Maturation of exported proteins in Escherichia coli . Requirement for energy, site and kinetics of processing; Pages JM et al.; Precursor forms of periplasmic and outer membrane proteins were accumulated in phenethyl-alcohol-treated cells in membrane fractions . After removal of phenethyl alcohol, maturation occurred in the absence but not in the presence of carbonylcyanide m-chlorophenylhydrazone (30 microM) . The site and kinetics of processing were investigated for OmpA, LamB and OmpF proteins and for maltose binding protein and TEM beta-lactamase . With regard to sites of processing, no fundamental difference between outer membrane and periplasmic proteins was observed . For maltose binding protein and TEM beta-lactamase, maturation, like that of outer membrane protein precursors, occurred in membrane fractions . Processing of pro-OmpA protein was about as fast as that of pro-LamB protein whereas pro-OmpF protein appeared to mature more slowly . While carbonylcyanide m-chlorophenylhydrazone (30 microM) prevented processing of all precursor forms, arsenate, which alters formation of ATP even when it was used at 1 mM, did not totally prevent maturation occurring . These results are discussed with regard to the biosynthesis and assembly of exported proteins.

Jpn J Antibiot, 1982 Jun, 35(6), 1562 - 6
{Spectinomycin therapy in combination with ampicillin in female patients with acute gonorrhea}; Masuda S et al.; Clinical effects of spectinomycin (Trobicin, 2 g . i.m., once a day) and ampicillin (1 g/day, p.o., in 4 divided doses, for 2 days) given in combination on acute gonorrhea in female patients were studied, and sensitivity of isolated gonococci to each of these antibiotics was determined . 1 . Clinical effects were evaluated by cultures of gonococci from discharge collected at 72 hours after spectinomycin administration and 12 hours after the last dose of ampicillin and both subjective and objective clinical findings . Among 20 cases of gonorrhea treated, 12 cases (60.0%) proved excellent in therapeutic results, 6 cases (30.0%) good, 1 case fair and 1 case produced no response . The success rate comprising excellent and good was 90.0% . The only side effect reported was pain of injection site in 1 case on 3 days after initiation of treatment, but no induration was noted . 2 . MICs of spectinomycin and ampicillin for gonococci collected from the 20 cases were determined . Spectinomycin showed MICs of 6.25 approximately 12.5 micrograms/ml against 10(8) CFU/ml and 3.13 approximately 12.5 micrograms/ml against 10(8) CFU/ml . These values were below the blood concentration obtained at 6 hours after intramuscular administration of 2 g of spectinomycin . Ampicillin had MICs of 0.1 approximately 25 micrograms/ml against 10(8) CFU/ml and 0.1 approximately 3.13 micrograms/ml against 10(6) CFU/ml . Five strains, MICs of which were 6.25 approximately 25 micrograms/ml against 10(8) CFU/ml, were beta-lactamase-producing.

Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3570 - 4
Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome; Wong EM et al.; We have isolated two mutant plasmid derivatives of ColE1 that exhibit temperature-sensitive replication properties . Both mutants have a normal copy number at 30 degrees C but increase their copy number 30- to 40-fold after a shift in temperature to 42 degrees C . A plasmid-encoded enzyme, beta-lactamase (penicillinase, EC 3.5.2.6), undergoes a 30- to 40-fold increase in specific activity concomitant with the increase in plasmid copy number . The copts phenotype of these mutants is not due to the synthesis of a temperature-sensitive polypeptide . Both mutations are located in an untranslated region of the plasmid genome encoding two overlapping transcripts involved in plasmid replication: a small transcript known as RNA I that acts as a negative control element in replication and a large transcript that has been characterized as the replication primer in vitro . The mutations alter the sequence encoding the primer but lie immediately 5' to the initiating nucleotide of RNA I, in the RNA I promoter region . The possibility that the temperature-dependent plasmid DNA amplification is a consequence of a temperature-sensitive RNA I promoter was tested by inserting the RNA I promoters from the wild-type and mutant plasmids into a plasmid in which galactokinase expression is dependent upon an exogenous promoter . These experiments demonstrate that the mutant promoters are not temperature-sensitive . Rather, the mutations may affect the secondary structure of the replication primer in a region important for RNA I interaction.

Can J Biochem, 1982 May, 60(5), 521 - 4
Expression of a cloned bovine growth hormone gene in Escherichia coli minicells; Rosner A et al.; The synthesis of polypeptides in Escherichia coli minicells, directed by a pBR322 plasmid and its derivative-carrying bovine growth hormone cDNA insert, was studied . Two polypeptides coded by the ampicillin-resistance (Apr) gene (32 000 and 28 000 daltons) and a tetracycline-resistance (Tcr) polypeptide (36 000 daltons) were identified by insertion inactivation . Two additional polypeptides of 37 000 and 34 000 daltons of as yet unknown function were detected in all extracts regardless of the presence of the Apr or Tcr genes in the plasmid . The pBR322-BGH recombinant plasmid coded for several novel polypeptides, among them one of 46 000 daltons, presumably a fused product of the BGH and beta-lactamase genes . This protein, however, was not secreted into the periplasmic space of the cells as was the beta-lactamase.

Br J Vener Dis, 1982 Apr, 58(2), 101 - 4
Treatment of pharyngeal gonorrhea due to beta-lactamase-producing gonococci; Lindberg M et al.; Between March 1978 and October 1980 seven patients with beta-lactamase-producing gonococci in the pharynx were treated wit spectinomycin of cefuroxime or both . One-day treatment with spectinomycin was effective in only one of six patients and with cefuroxime in only one of four patients . Prolonged treatment with cefuroxime was successful in all five cases so treated.

Proc Natl Acad Sci U S A, 1982 Apr, 79(8), 2475 - 9
Production in Escherichia coli of biologically active secretin, a gastrointestinal hormone; Suzuki M et al.; A synthetic gene for porcine secretin was ligated with Pst I-cleaved pBR322 by its flanking synthetic Pst I linkers to produce a fused protein consisting of an amino-terminal portion of beta-lactamase and an entire secretin molecule . The hybrid plasmid was transferred into competent Escherichia coli cells . The plasmids, which were proved in our investigation to contain the secretin gene in the desired orientation, were then screened for the production of secretin . This was shown by radioimmunoassay and gel electrophoretic analysis of the polypeptides that were synthesized in E . coli minicells transformed with the hybrid plasmids . Secretin so produced, whose carboxy terminal residue may not be amidated in contrast with natural porcine secretin, showed the same activity in stimulating pancreatic secretion in a bioassay with anesthetized rats as does natural secretin.

J Bacteriol, 1982 Apr, 150(1), 269 - 76
Comparison of transcription of beta-lactamase genes specified by various ampicillin transposons; Yamamoto T et al.; The beta-lactamase gene from four kinds of ampicillin transposons, Tn2601, Tn3, Tn2602 and Tn1, specifying the type I (or TEM type, alternatively) beta-lactamase was cloned onto plasmid pACYC184, and the level of in vivo transcription from each beta-lactamase gene was determined by DNA-RNA hybridization . Type I beta-lactamase is very uniform enzymologically, but heterogeneous in absolute levels of enzyme activity . The results demonstrated that the heterogeneity can be explained by the efficiency of transcription of each beta-lactamase gene, suggesting a difference in its promoter efficiency . A comparison of the levels of transcription of the beta-lactamase gene and the whole ampicillin transposon suggested that the beta-lactamase gene has the strongest promoter all of the genes in the ampicillin transposon.

Antimicrob Agents Chemother, 1982 Mar, 21(3), 506 - 8
beta-Lactamases of Branhamella catarrhalis and their inhibition by clavulanic acid; Farmer T et al.; Three of four clinical isolates of Branhamella catarrhalis from Belgium produced a beta-lactamase identical to enzymes previously reported to occur in French and British isolates of this organism . One strain, however, produced a new type of beta-lactamase . Both beta-lactamase types were readily inhibited by low concentrations of clavulanic acid.

Proc Natl Acad Sci U S A, 1982 Mar, 79(5), 1466 - 8
Use of different tRNASer isoacceptor species in vitro to discriminate between the expression of plasmid genes; Cenatiempo Y et al.; A simplified translation system coupled to DNA transcription that involves assaying the synthesis of the first dipeptide of a gene product has been described recently {Robakis, N., Meza-Basso, L., Brot, N . & Weissbach, H . (1981) Proc . Natl . Acad . Sci . USA 78, 4261--4264} . Using this dipeptide system, we have investigated the expression of genes carried on plasmids coding for beta-lactamase, ribosomal protein L12, and the chloroplast large subunit (LS) of ribulosebisphosphate carboxylase (RbuBPCase) . Although all three nascent gene products begin with the sequence fMet-Ser, the formation of fMet-Ser can be used to distinguish between the synthesis of beta-lactamase and either L12 or the LS of RbuBPCase by using different serine isoacceptor tRNA species . In beta-lactamase, the serine codon is AGU, which utilizes the serine isoacceptor species tRNASer3; in L12 and the LS of RbuBPCase, the serine codewords are UCU and UCA, respectively, both of which are recognized by the serine isoacceptor species tRNASer1 . By using either pure tRNASer1 or pure tRNASer3, the expression of each gene can be quantitated . In this system, guanosine-5'-diphosphate-3'-diphosphate inhibits the expression of the beta-lactamase and L12 genes but stimulates the synthesis of the LS . In addition, the ratio of fMet-Ser/fMet-Ala (L12/L10) synthesized was about 1 as compared with the ratio of 4 that has been obtained previously in vivo or in vitro protein-synthesizing systems in which the entire gene product was measured.

Ital J Biochem, 1982 Jan-Feb, 31(1), 1 - 7
Do intermolecular association phenomena occur in B . cereus beta-lactamase I?
Amicosante G, Crifo C, Strom R.
Although at fixed enzyme concentration the hydrolysis of cephaloridine by B . cereus beta-lactamase I followed apparently classical Michaelis-Menten steady-state kinetics, the values of kcat and of Km depended linearly, in the absence of added non-enzymatic proteins, on the absolute enzyme concentration . In the presence of gelatin, this dependence was abolished; under these conditions, however, the pseudo-first order kinetic rate constants of inactivation by Zn2+ ions exhibited a direct dependence on enzyme concentration and an inverse one on Zn2+ ions concentration . These results can be interpreted as indicating that intermolecular association phenomena play a role in determining the catalytic properties of the enzyme.

EMBO J, 1982, 1(11), 1411 - 6
Identification of a novel ampC beta-lactamase promoter in a clinical isolate of Escherichia coli; Olsson O et al.; A clinical strain of Escherichia coli, C16, that overproduces the ampC beta-lactamase was isolated . A 203-bp DNA segment from this strain, including the promoter and attenuator region of the ampC structural gene, was sequenced . A comparison with the corresponding sequence of E . coli K12 revealed four base pair differences between the ampC segments from these strains . DNA sequence data and in vitro transcription indicated that the ampC promoter in the clinical isolate was displaced 5 bp upstream of the promotor defined in the E . coli K12 strain . Like the ampC gene of E . coli K12, the ampC gene from the clinical isolate was metabolically regulated . However, the increase in the specific amount of beta-lactamase relative to the increase in specific growth rate was much higher in the clinical isolate . These data imply that the growth rate-dependent anti-termination acting on the ampC attenuator in vivo is more pronounced in the clinical E . coli isolate than in E . coli K12 . A possible molecular mechanism for this is discussed.

EMBO J, 1982, 1(7), 875 - 81
Sequence elements determining ampC promoter strength in E . coli; Jaurin B et al.; A number of spontaneous up-promoter mutations have been isolated in the ampC beta-lactamase gene of Escherichia coli . The mutants were analyzed by DNA sequencing, and the level of ampC gene expression was determined . Six mutants with a 21-fold increase in promoter strength compared with the wild-type were mutated in the -35 promoter region from TTGTCA to the consensus sequence TTGACA . The -10 region sequence TACAAT was mutated to the consensus sequence TATAAT in three mutants exhibiting an ampC promoter seven times stronger than the wild-type . We have previously described a 1-bp insertion mutant ( Jaurin et al., 1981) that changes the inter-region distance to the consensus 17 bp . Thus, all the up-mutations found in the ampC promoter represent corrections of the three major discrepancies between the ampC promoter and the consensus E . coli promoter . We conclude that the three consensus elements of E . coli promoters, the -35 and -10 regions and an optimal inter-region distance of 17 bp, are the main elements determining the promoter strength.

Mol Gen Genet, 1982, 187(2), 187 - 94
Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660; Chiang SJ et al.; Intermolecular transposition of Tn2660 into pCR1 was measured at 30 degrees C in recA- and recA+ hosts as between 2.6 and 5.5 X 10(-3), a similar value to that previously found for Tn3 . No cointegrate structures were found under conditions where 10(4) transposition events occurred . Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10(-3) and 10(-4) when a mutant Tn2660 (resulting in the synthesis of a temperature-sensitive beta-lactamase) was present in the recipient plasmid . Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted . Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.

Mol Gen Genet, 1982, 185(2), 302 - 10
Analysis of lambda receptor and beta-lactamase synthesis and export using cloned genes in a minicell system; Clement JM et al.; We have cloned lamB, the gene for lambda receptor (an outer membrane protein), on a small plasmid which also carries the gene for beta-lactamase (a periplasmic protein) . We have identified a promoter in the region of malK, the gene immediately preceding lamB, which is active in minicells but relatively inactive in vitro . Using a minicell system, we have found that both lambda receptor and beta-lactamase are made as full length precursors which are subsequently processed . We also show that the lambda receptor precursor can be exported to the outer membrane before it is processed . Mature beta-lactamase is found only in the periplasm, suggesting that processing may be a requirement for export to the periplasm.

Proc Natl Acad Sci U S A, 1982 Jan, 79(2), 520 - 4
Isolation of transforming DNA by cosmid rescue; Lund T et al.; A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences . DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant . Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid . Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids . This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.

Nucleic Acids Res, 1981 Dec 21, 9(24), 6669 - 88
Albumin is encoded by 2 messenger RNAs in Xenopus laevis; Schoenberg DR; A cDNA clone library was prepared from liver poly(A) RNA pf non-estrogenized Xenopus laevis . Albumin coding sequences were screened by hybridization to a cDNA prepared from poly(A) RNA enriched by sucrose density gradient centrifugation, and by a sensitive solid-phase radioimmunoassay to detect clones that contain templates for albumin antigenic determinants . Nine clones were obtained by this approach, and all but one have the cDNA inserted in phase with the beta-lactamase gene of pBR322 . Mapping of these clones with restriction endonucleases yielded 2 distinct patterns, suggestive of heterogeneity in the coding sequences . This was confirmed by heteroduplex analyses of hybrids formed between clones representative of each of the 2 classes . Both classes of albumin cDNA clones were used to select mRNAs of the same size (2.3kb) that code for peptides that are indistinguishable by SDS gel electrophoresis . Examination of the organization of the albumin genes by blot hybridization of the cDNA clones to restriction fragments of Xenopus DNA failed to detect any differences at the genomic level . The considerable diversity of the albumin cDNAs is suggestive of a multiplicity of albumin genes, rather than differential processing of a common precursor RNA.

J Bacteriol, 1981 Dec, 148(3), 745 - 52
Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells; Gomez-Eichelmann MC; The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied . Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200% . Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent . The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant . The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells . These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.

Tubercle, 1981 Dec, 62(4), 289 - 93
Isoelectric focusing studies on Mycobacterium chelonei; Sparks J et al.; Analytical IEF has been used to give a direct visual comparison of beta-lactamases, down to extremely low levels, from very crude intracellular preparations . Identity and non-identity of strains could be proved by the pattern of bands produced by their beta-lactamases . Mycobacterial species gave a variety of beta-lactamase patterns . Identity was established between some strains of M . chelonei . IEF distinguished between enzymes within both the chelonei and abscessus sub-species that could not be differentiated by other methods . This technique could provide a means of identifying the source of a M . chelonei infection.

Br J Vener Dis, 1981 Dec, 57(6), 363 - 6
Sexually transmitted diseases in Sabah and Sarawak; Catterall RD; Despite being part of one of the few remaining primitive areas of the world, both Sabah and Sarawak are provided with adequate, though simple, urban and rural general medical services . At present no reliable data on the incidence of sexually transmitted diseases in these areas have been collected and no organised treatment services are available . Gonorrhoea appears to be the commonest notifiable infectious disease in Sarawak, and beta-lactamase-producing strains have been isolated . Because of the rapidly expanding economy and the encouragement of the tourist trade, sexually transmitted disease is likely to prove an increasing problem, for which a specialised service for diagnosis and treatment is badly needed.

J Med Chem, 1981 Dec, 24(12), 1531 - 4
Synthesis and beta-lactamase inhibitory properties of 2 beta-(chloromethyl)-2 alpha-methylpenam-3 alpha-carboxylic acid 1,1-dioxide; Gottstein WJ et al.; Potassium 2 beta-(chloromethyl)-2 alpha-methylpenam-3 alpha-carboxylate 1,1-dioxide (BL-P2013) and its pivaloyloxymethyl ester were prepared by the conversion of 6-aminopenicillanic acid to p-nitrobenzyl 6 alpha-bromo-2,2-dimethylpenam-3 alpha-carboxylate 1-oxide, which was rearranged with benzoyl chloride and quinoline to p-nitrobenzyl 6 alpha-bromo-2 beta-(chloromethyl)-2 alpha-methylpenam-3 alpha-carboxylate in 65% yield . Oxidation and catalytic hydrogenation afforded BL-P2013, which was found to be a potent inhibitor of various bacterial beta-lactamases and has been found to protect amoxicillin from beta-lactamases in both in vitro and in vivo systems.

Biochem J, 1981 Oct 1, 199(1), 137 - 43
Interactions between non-classical beta-lactam compounds and the beta-lactamases of Actinomadura R39 and Streptomyces albus G; Kelly JA et al.; 6-Aminopenicillanic acid, 7-aminocephalosporanic acid, mecillinam and quinacillin have varying substrate activities for both the R39 beta-lactamase (excreted by Actinomadura R39) and the G beta-lactamase (excreted by Streptomyces albus G) . Cefoxitin and quinacillin sulphone are not recognized by the G beta-lactamase and are weak inactivators of the R39 beta-lactamase . N-Formimidoylthienamycin is a poor substrate for the G beta-lactamase and a potent inactivator of the R39 beta-lactamase . The high value of the bimolecular rate constant for enzyme inactivation is mainly due to a very low dissociation constant (1 microM) . Clavulanate is an inactivator of both G and R39 beta-lactamases . The reaction with this latter enzyme is a branched pathway where normal turnover and permanent enzyme inactivation occur concomitantly . Between 28 and 43 molecules of clavulanate are hydrolysed before one of them has the opportunity to inactivate one molecule of enzyme.

Proc Natl Acad Sci U S A, 1981 Sep, 78(9), 5396 - 400
Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli; Daniels CJ et al.; The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space . The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP) . The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential . Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts . Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium . These results suggest a role for membrane potential in the secretion of periplasmic proteins . A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence within the membrane, thus promoting processing and secretion.

Infect Immun, 1981 Aug, 33(2), 632 - 5
Extracellular enzymes of Legionella pneumophila; Thorpe TC et al.; All strains of Legionella pneumophila tested produced detectable levels of extracellular protease, phosphatase, lipase, deoxyribonuclease, ribonuclease, and beta-lactamase activity . Weak starch hydrolysis was also demonstrated for all strains . Elastase, collagenase, phospholipase C, hyaluronidase, chondroitinase, neuraminidase, or coagulase were not detected in any of these laboratory-maintained strains.

J Bacteriol, 1981 Aug, 147(2), 578 - 88
A mutant defective in partitioning of composite plasmid Rms201; Ike Y et al.; Escherichia coli harboring mutant plasmids defective in maintenance stability (from the conjugative plasmid Rms201) showed a wide distribution of ampicillin resistance levels, as well as increased frequency of plasmid loss from the cell . The amounts of covalently closed circular deoxyribonucleic acid of mutant plasmid Rms268 and parental plasmid Rms201 per chromosome were 5.3 and 6.1%, respectively . The beta-lactamase activities of strains W3630(Rms268) and W3630(Rms201) were 0.56 and 0.44 U/mg of protein, respectively . Frequency of plasmid loss from W3630(Rms268) was about 0.8 to 1.2% per cell generation, 100 times more than that of the wild-type strain . Ampicillin resistance levels of the colonies harboring the mutant plasmid showed a wide distribution, from low (100 micrograms/ml) to high (1,600 micrograms/ml) . A miniplasmid (pMS268) with a mass of 7 X 10(6) daltons and encoding ampicillin resistance was isolated from Rms268 . Frequency of pMS268 loss from W3630(pMS268) was about 0.8 to 1.9% per cell generation . W3630(pMS268) also showed a wide range of distribution in the levels of ampicillin resistance . These results indicated that the copies of Rms268 in E . coli did not segregate evenly between daughter cells at cell division and that the gene involved was located on the miniplasmid.

Nature, 1981 Jul 16, 292(5820), 269 - 71
Recombination between short DNA homologies causes tandem duplication; Edlund T et al.; The ampC gene of Escherichia coli K-12 codes for a beta-lactamase which can hydrolyse the beta-lactam ring of ampicillin . Ampicillin resistance is strictly related to ampC gene copy number thus we have been able to isolate ampicillin-resistant mutants carrying multiple ampC repeats . We have isolated on a plasmid a segment of chromosomal DNA carrying multiple ampC repeats, and compared the nucleotide sequence of the region joining repeat units to the sequence of the DNA segments that fused to create the joint . The fusion had occurred within a 12-base pair (bp) sequence of perfect homology . We suggest that recombination between randomly occurring short homologies (12-13-bp long), could be a general mechanism to generate tandem duplications in the size range of 10 kilobases (kb).

Cell, 1981 Jul, 25(1), 151 - 7
Different exported proteins in E . coli show differences in the temporal mode of processing in vivo; Josefsson LG et al.; A number of exported proteins in E . coli, both periplasmic proteins and proteins of the outer membrane, were examined to determine when removal of the "signal sequence" occurs in vivo . One protein was processed entirely cotranslationally (amp C beta-lactamase) and one was processed entirely post-translationally (TEM beta-lactamase) . The others (maltose-binding protein, arabinose-binding protein, omp A protein, lam B protein and alkaline phosphatase) showed both modes of processing, although the amount of cotranslational processing varied considerably among the individual proteins of this class . When processing occurred cotranslationally, the proteolytic removal of the "signal" was a late event . For four of the proteins studied, processing was initiated only after the polypeptides had been elongated to approximately 80% of their full length.

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