Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system.

An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage. A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9. A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated. From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105. It was used to prepare a lysate of the phi 105dspo0A12 phage. Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out. The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells. These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene. Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca. 10(-3) in the sporulation medium. This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate.     

Deletion mutants of temperate Bacillus subtilis bacteriophage phi105.

Summary Six deletion mutants of temperate Bacillus subtilis phage 105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55–70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55–65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65–70% from the left end.By electronmicroscopy of partially denatured 105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55–65% DNA segment.     

The active site of the P99 beta-lactamase from Enterobacter cloacae.

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.     

Evidence for circular permutation of the prophage genome of Bacillus subtilis bacteriophage phi 105.

Analysis of DNA extracted from Bacillus subtilis lysogenic for bacteriophage phi 105 was performed by restriction endonuclease digestion and Southern hybridization using mature phi 105 DNA as a probe. The data revealed that the phi 105 prophage is circularly permuted. Digests using the enzymes EcoRI, SmaI, PstI, and HindIII localized the bacteriophage attachment site (att) to a region 63.4 to 65.7% from the left end of the mature bacteriophage genome. The phi 105 att site-containing SmaI C, PstI J, and HindIII L fragments were not present in digests of phi 105 prophage DNA. phi 105-homologous "junction" fragments were visualized by probing digests of prophage DNA with the purified PstI J fragment isolated from the mature bacteriophage genome. The excision of the phi 105 prophage was detected by observing the appearance of the mature PstI J fragment and the concomitant disappearance of a junction fragment during the course of prophage induction.     

Molecular modeling of Henry-Michaelis and acyl-enzyme complexes between imipenem and Enterobacter cloacae P99 beta-lactamase

We report a molecular-mechanics (AMBER*) study on the Henry-Michaelis complex and the corresponding acyl-enzyme adduct formed between imipenem (1), a transient inhibitor of beta-lactamases, and Enterobacter cloacae P99, a class C-beta-lactamase. We have examined the influence of the structural configuration of the functional groups in the substrate on their three-dimensional (3D) arrangement at the active site, which was compared with those adopted by typical penicillins and cephalosporins. Our results confirm that the carboxy group of the antibiotic plays a prominent role in the binding of the substrate to the active site, and that it activates Ser64 through interaction with the phenolic OH group of Tyr150. The binding of imipenem to E. cloacae P99 increases the distance between Tyr150 and Ser64 due to the presence of a hydrophobic Me group in the (R)-1-hydroxyethyl substituent at C(6). This, together with the 3D arrangement of its carboxy group, leads to an interaction with the active site in a manner that hinders H+ exchange between the nucleophile in Ser64 and its basic activator, the phenolic group of Tyr150.     

The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99

The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.     

Common mechanism of ampC beta-lactamase induction in enterobacteria: regulation of the cloned Enterobacter cloacae P99 beta-lactamase gene.

Expression of the chromosomal beta-lactamase from the ampC gene in inducible in both Enterobacter cloacae and Citrobacter freundii. Cloning of ampC as well as its regulatory gene, ampR, from E. cloacae P99 revealed a gene organization indentical to that of C. freundii in the corresponding region. Although almost no similarities could be found between the restriction maps of ampC and ampR in the two species, the genes cross-hybridize. Also, both ampR gene products have a size of about 31,000. The regulatory features of E. cloacae beta-lactamase induction are very similar to those in C. freundii, i.e., beta-lactamase synthesis is repressed by AmpR in the absence, and stimulated in the presence, of inducer. The AmpR function can be transcomplemented between the two species, but there are quantitative regulatory aberrations in such hybrids, in contrast to the total complementation obtained within each system. These results suggest that the mechanism of beta-lactamase induction is the same in E. cloacae, C. freundii, and other gram-negative bacteria with inducible chromosomal beta-lactamase expression.     

Mechanism of reaction of acyl phosph(on)ates with the beta-lactamase of Enterobacter cloacae P99

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.     

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.     

Co-expression of a prophage system and a plasmid system in Bacillus subtilis

A dual expression system for overexpressing two proteins by a single cell strain has been developed in Bacillus subtilis. This dual expression system combines the phi105MU331 prophage system and a plasmid system within a single cell. Protein expression by the prophage system is heat inducible, while that of the plasmid system is constitutive. Three candidate genes, BPN, BT, and amyE, all of Bacillus origin, were used as test models. Seven strains (BPN, BT, AMY, BS168K, MU331K, BPNK, and BTK) were constructed to investigate the influences of the prophage system and the plasmid system on each other, and to compare the efficiency of the individual expression systems with that of the dual expression system. Individually, the yield of the plasmid system is higher than that of the prophage system, which could be attributed to the constitutive nature of the expression of the plasmid system. Nonetheless, for the dual expression strains, the expression of two enzymes in a single fermentation run can reduce costs in facilities, manpower, and utilities. Fed-batch fermentation of BPNK strains confirmed the feasibility of applying this dual expression system in industrial-scale production.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

Expression of AmpC beta-lactamase in Enterobacter cloacae isolated from retail ground beef, cattle farm and processing facilities

AIMS: To better understand antibiotic resistance of Enterobacter cloacae isolates originated from food animals, the phenotypic and genotypic resistance of Ent. cloacae isolates from retail ground beef, cattle farm, processing facilities and clinical settings were investigated. METHODS AND RESULTS: The ampC, ampD and ampR genes in the isolates were sequenced and analysed. beta-Lactamase activities and beta-lactamase profiles of the isolates were analysed by the enzymatic hydrolysis of nitrocefin and isoelectric focussing, respectively. The ampC gene of the Ent. cloacae isolate was cloned and transformed into Escherichia coli strains. The genomic DNA profiles of Ent. cloacae isolates were analysed by using pulse field gel electrophoresis (PFGE). Mutation at one residue (Val-54-->Ile) in the AmpR amino acid sequence was consistently found in Ent. cloacae isolates that were resistant to a broadspectrum of beta-lactam agents. The enzyme activity in the isolates was induced by cefoxitin. The pI (isoelectric point) of the enzymes produced by the test strains ranged from 8.4 to 8.9. Cloning of ampC gene of the Ent. cloacae isolate conferred the resistance to ampicillin, cephalothin and amoxicillin in recipient E. coli strains. One recipient of E. coli O157:H7 strain additionally acquired resistance to ceftiofur. The genomic analysis of Ent. cloacae isolates by PFGE showed that the isolates from various sources were genetically unrelated. CONCLUSIONS: The spread of diverse clones of AmpC-producing Ent. cloacae occurred in the ecosystem and retail products. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggested that AmpC-producing Ent. cloacae could be a contributor in spreading beta-lactamase genes in farm environments and food processing environments.     

The beta-lactamase of Enterobacter cloacae P99. Chemical properties, N-terminal sequence and interaction with 6 beta-halogenopenicillanates.

The beta-lactamase of Enterobacter cloacae P99 consists of one polypeptide chain of Mr 39000 devoid of disulphide bridges and free thiol groups. It contains an unusually high proportion of tyrosine and tryptophan. The N-terminal sequence exhibits overlaps with the tryptic peptide obtained after labelling the active site with 6 beta-iodopenicillanate. The active-site serine residue is at position 64. The homology with the chromosomal beta-lactamase of Escherichia coli K 12 (ampC gene) is lower within the 25 residues of the N-terminal portion than around the active-site serine residue. The P99 beta-lactamase is inactivated by 6 beta-bromo- and 6 beta-iodo-penicillanate, with a second-order rate constant of 110-140M-1 X s-1 at 30 degrees C and pH 7.0, a value that is much lower than that observed with class-A beta-lactamases.     

Interaction of the Bacillus subtilis phage phi 105 repressor DNA: a genetic analysis.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.     

Location of the Bacillus subtilis temperate bacteriophage phi 105 attP attachment site.

Chromosomal DNAs of lysogens of phi 105 and phi 105 DI:1t were digested with restriction enzymes EcoRI and HpaI and were probed with nick-translated mature phi 105 DNA. Altered bacteriophage-specific bands in the lysogens were detected, indicating that the phage integrates into the host chromosome at a single site, probably via a Campbell-type circular intermediate. The phage attachment site is centrally located in the phage genome and lies between the phage immunity region and the nonessential deletable region of phi 105.     

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.     

Reorienting and expanding the physical map of temperate Bacillus subtilis bacteriophage phi 105.

During the course of extending the physical map of Bacillus subtilis temperate bacteriophage phi 105 to include SstI, XhoI, and HpaI, we recognized that the previous physical map for EcoRI was incorrect. The new enzyme maps were determined by single, double, and partial enzyme digestions, redigestion of purified phage fragments, end joint analysis, and DNA-DNA hybridization. The EcoRI physical map was corrected by double digestion of isolated fragments, DNA hybridization, and physical mapping by partial digestion of end-labeled fragments. EcoRI fragment G was repositioned to give the order D-G-I-E-B-H-F-C.