Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [³H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [³H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [³H]-benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.     

Autolysins are direct involved in the bactericidal effect caused by penicillin in wild type and in tolerant pneumococci

The two pneumococcal autolytic enzymes (an N-acetylmuramoyl-L-alanine amidase and an endo-beta-1,4-N-acetylglucosaminidase) are directly involved in the penicillin-induced killing of Streptococcus pneumoniae. The activity of these lytic enzymes was efficiently controlled in tolerant mutants under physiological conditions.     

Penicillin-binding proteins of pathogenic Escherichia coli strains

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [³H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by β-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.     

Penicillin-binding protein 4 of Escherichia coli shows a novel type of primary structure among penicillin-interacting proteins

The nucleotide sequence of a 1884 bp DNA fragment of E. coli, carrying the gene dacB, was determined. The DNA codes for penicillin-binding protein 4 (PBP4), an enzyme of 477 amino acids, being involved as a DD-carboxypeptidase-endopeptidase in murein metabolism. The enzyme is translated with a cleavable signal peptide of 20 amino acids, which was verified by sequencing the amino-terminus of the isolated protein. The characteristic active-site fingerprints SXXK, SXN and KTG of class A beta-lactamases and penicillin-binding proteins were located in the sequence. On the basis of amino acid alignments we propose, that PBP4 and class A beta-lactamases share a common evolutionary origin but PBP4 has acquired an additional domain of 188 amino acids in the region between the SXXK and SXN elements.     

Methicillin-resistant strains of Staphylococcus aureus; presence of identical additional penicillin-binding protein in all strains examined

Abstract The methicillin-resistant strain of Staphylococcus aureus MR-1 previously reported to possess a penicillin-binding protein 3 (PBP 3) with a decreased affinity for β-lactam antibiotics was re-examined and, in common with other resistant strains, found to contain an additional PBP (PBP 2'). Expression of the additional protein, which has a very low affinity for β-lactams, was not influenced by temperature or osmolarity of the medium in contrast with strains examined previously. It was the only PBP still available to bind radioactive β-lactams and therefore still active enzymically when strain MR-1 was grown in the presence of concentrations of β-lactam antibiotics sufficient to kill sensitive strains of S. aureus . Penicillin-peptides derived by partial proteolysis of PBP 2'-penicillin complexes of MR-1 and 3 other methicillin-resistant strains appeared to be identical and different from the penicillin-peptides derived from PBP 1, PBP 2 and PBP 3, each of which gave rise to a unique series of peptides containing covalently-bound penicillin.     

Interaction between monobactams and model d-alanyl-d-alanine-cleaving peptidases

Abstract Several monobactams reacted with the serine dd -peptidases of Streptomyces R61 and Actinomadura R39 in a manner similar to that of bicyclic penicillins and cephalosporins. The dissociation constants of the Michaelis complexes formed between the R61 enzyme and sulfazecin (32 μM) and between the R39 peptidase and SQ 26324 (0.35 μM) had the lowest values ever observed with any β-lactam compound, suggesting an excellent fit of these two monobactams with the active sites of the respective enzymes. Azthreonam had a very poor inactivating potency, confirming its high selective reactivity towards the penicillin binding protein No. 3 of Escherichia coli . The Zn²⁺ dd -peptidase (from Streptomyces albus G) had a high intrinsic resistance to β-lactam compounds whether they possessed a mono- or a bicyclic structure.     

Endogenous ADP-ribosylation of proteins in Mycobacterium smegmatis.

Endogenous ADP-ribosylation of two proteins with molecular weights of 30,000 (30K) and 80,000 (80K) was detected in cell extracts of Mycobacterium smegmatis. Modification of these proteins was enzymatic. The ADP-ribose bound to 30K was removed by HgCl2 but not by NH2OH, suggesting the modification of a cysteine residue. The ADP-ribose bound to 80K was not removed by either HgCl2 or NH2OH, which is consistent with the modification of an asparagine residue. ADP-ribosylation of 80K appeared to be reversible.     

Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine beta-lyase. With this new host-vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein-PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL-SKL produced in H. polymorpha displays normal enzymatic activities.     

Involvement of O8-antigen in altering β-lactam antibiotic susceptibilities in Escherichia coli

In spite of being dispensable, O-antigens are believed to facilitate various cellular processes and alter antibiotic sensitivities. Escherichia coli K-12 (CS109) strains are lacking in O-antigens and are reported to be sensitive to antibiotics. To our surprise, E. coli 2443 (expressing O8-antigen) manifested two- to fourfold higher sensitivities toward penicillin and its derivatives than strain CS109. However, sensitivities toward other structurally unrelated antibiotics remained unchanged. To understand the rationale behind such observations, we replaced the rfb locus of strain 2443 with that of E. coli K-12. The β-lactam sensitivities of 2443 cells with replaced rfb locus appeared to be identical to those for CS109. Therefore, it is quite reasonable to hypothesize the possible involvement of O8-antigen in β-lactam sensitization.     

NMR spectrometric assay for determining enzymatic hydrolysis of β-lactam antibiotics with bacteria in aqueous solution

Abstract An application of a nuclear magnetic resonance (NMR) spectrometer for the measurement of β-lactamase activity in clinical material containing bacteria is presented. By means of proton (¹H)-NMR, it was easy to measure quantitatively β-lactamase activity in human bacteriuria, without performing any such pretreatment as isolation of bacteria or extraction of crude enzymes and without preparing special reagents for the detection. This is the first report on the application of ¹H-NMR analysis of structural changes for determining hydrolysis of β-lactam antibiotics with β-lactamase-producing bacteria in aqueous solution.     

Binding sites of sorbed uranyl ion in the cell wall of Mycobacterium smegmatis

Abstract A study of cell-wall site interaction of uranyl ion adsorbed by non-proliferative suspensions of Mycobacterium smegmatis at pH 1 has been carried out using extracts of arabinogalactan-peptidoglycan and phospholipids obtained from whole cells treated under sorption conditions. Evidence for binding of UO₂²⁺ by constituent P-lipids was provided by comparative ³¹P-NMR and IR spectroscopic measurements of the isolated wall fractions and of the model complex uranyl-phosphatidyl inositol.     

Identification and characterization of a diamide sensitive mutant of Mycobacterium smegmatis

A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc(2)155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.     

Resistance heterogeneity in methicillin-resistant Staphylococcus aureus

Abstract A striking feature of methicillin resistance in Staphylococcus aureus is the considerable heterogeneity of expression of resistance by cells in clonal populations: some are sensitive (or almost so), others are highly resistant, and others show intermediate resistance to the antibiotic. Subclones generally are also heterogeneous, suggesting variable inheritance or control of expression of resistance.
The degree of heterogeneity and mean resistance is influenced by environmental parameters: temperature, osmolality, pH, light, anaerobiosis, chelating agents and metal ions, and prior exposure to β-lactam antibiotics.     

Cytokinin-binding proteins

This article is focused on the modalities of reception of cytokinins which remain largely unknown. It summarizes the main steps of the different protocols used to study cytokinin-binding proteins (CBPs). We place emphasis on the significance and specificity of the detection according to the properties of the probes used: radioactive or photoreactive cytokinins, fluorescent anticytokinins, anti-idiotype antibodies. The purification procedures are also examined. The cellular localisation and the putative physiological roles of the numerous and different CBPs found are considered. The interest of genetic and molecular studies is discussed.     

The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.     

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over‐express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non‐pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.     

Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI>8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.     

Application of cross-linked enzyme aggregates of Bacillus badius penicillin G acylase for the production of 6-aminopenicillanic acid

AIMS: Optimization of 6-aminopenicillanic acid (6-APA) production using cross-linked enzyme aggregates (CLEA) of Bacillus badius penicillin G acylase (PAC). METHODS AND RESULTS: CLEA-PAC was prepared using purified/partially purified PAC with phenylacetic acid as active-site blocking agent and glutaraldehyde as cross-linker. Conversion of penicillin G to 6-APA by CLEA-PAC was optimized using response surface methodology (RSM) (central composite rotatable design) consisting of a three-factor-two-level pattern with 20 experimental runs. CONCLUSION: Nearly, 80% of immobilization yield was obtained when partially purified enzyme was used for the preparation of CLEA-PAC. Quantitative conversion of penicillin G to 6-APA was observed within 60 min and the CLEA-PAC was reusable for 20 repeated cycles with 100% retention of enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The faster conversion of penicillin G to 6-APA by CLEA-PAC and efficient reusability holds a strong potential for the industrial application.     

Spider acetylcholine binding proteins: An alternative model to study the interaction between insect nAChRs and neonicotinoids

Acetylcholine binding proteins (AChBPs) are homologs of extracellular domains of nicotinic acetylcholine receptors (nAChRs) and serve as models for studies on nAChRs. Particularly, studies on invertebrate nAChRs that are limited due to difficulties in their heterologous expression have benefitted from the discovery of AChBPs. Thus far, AChBPs have been characterized only in aquatic mollusks, which have shown low sensitivity to neonicotinoids, the insecticides targeting insect nAChRs. However, AChBPs were also found in spiders based on the sequence and tissue expression analysis. Here, we report five AChBP subunits in Pardosa pseudoannulata, a predator enemy against rice insect pests. Spider AChBP subunits shared higher sequence similarities with nAChR subunits of both insects and mammals compared with mollusk AChBP subunits. The AChBP1 subunit of P. pseudoannulata (Pp-AChBP) was then expressed in Sf9 cells. The Ls-AChBP from Lymnaea stagnalis was also expressed for comparison. In both AChBPs, one ligand site per subunit was present at each interface between two adjacent subunits. Neonicotinoids had higher affinities (7.9–18.4 times based on K_d or K_i values) for Pp-AChBP than for Ls-AChBP, although epibatidine and α-bungarotoxin showed higher affinities for Ls-AChBP. These results indicate that spider AChBP could be used as an alternative model to study the interaction between insect nAChRs and neonicotinoids.