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The Streptomyces clavuligerus genome consists in a linear chromosome of about 6.7 Mb and four plasmids (pSCL1 to pSCL4), the latter one of 1.8 Mb. Deletion of pSCL4, results in viable mutants with high instability in the chromosome arms, which may lead to chromosome circularisation. Transcriptomic and proteomic studies comparing different mutants with the wild-type strain improved our knowledge on the biosynthesis and regulation of clavulanic acid, cephamycin C and holomycin. Additional knowledge has been obtained on the SARP-type CcaR activator and the network of connections with other regulators (Brp, AreB, AdpA, BldG, RelA) controlling ccaR expression. The transcriptional pattern of the cephamycin and clavulanic acid clusters is supported by the binding of CcaR to different promoters and confirmed that ClaR is a CcaR-dependent activator that controls the late steps of clavulanic biosynthesis. Metabolomic studies allowed the detection of new metabolites produced by S. clavuligerus such as naringenin, desferroxamines, several N-acyl tunicamycins, the terpenes carveol and cuminyl alcohol or bafilomycin J. Heterologous expression of S. clavuligerus terpene synthases resulted in the formation of no less than 15 different terpenes, although none of them was detected in S. clavuligerus culture broth. In summary, application of the Omic tools results in a better understanding of the molecular biology of S. clavuligerus, that allows the use of this strain as an industrial actinobacterial platform and helps to improve CA production.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

Purpose: Optimal treatment regimens are yet to be established for carbapenemase-producing Enterobacteriaceae (CPE). We assessed the in vitro efficacy of meropenem (MEM) and cefmetazole (CMZ) combin...

Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem–cefoxitin combination and doripenem–tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem–cefoxitin treatment and by 35.54% after doripenem–tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem–cefoxitin and doripenem–tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.

Background: Apparently healthy individuals could serve as reservoirs and disseminators of extendedspectrum cephalosporin (ESC)- and cephamycin (cefoxitin, FOX)-resistant, and extended-spectrum β-lactamase-producing (ESBL-P) Escherichia coli which jeopardizes antibacterial therapy thereby posing a threat to the health of infected individuals/carriers. Objectives: This study aimed to screen healthy asymptomatic students in the University of Nigeria, Nsukka (UNN) as potential reservoirs of ESC- and FOX-resistant and ESBL-P E. coli and to determine the antibacterial resistance profile. Materials and Methods: Anal swabs were collected from 190 randomly selected healthy asymptomatic students of both genders in UNN between March and July 2018. ESC-resistant E. coli was isolated using MacConkey agar with 2 µg/mL ceftazidime. ESBL production was assessed by combination disc method while cephamycin resistance was determined using cefoxitin disc screening. Phenotypic resistance of the isolates was determined using disc diffusion method. Results: Out of 190 samples, 20 (10.2%) demonstrated growth. Of these, 6 (30%) were FOX resistant (putative AmpC-producers) but none produced ESBL. The resistance of the isolates was 100% to ampicillin (AMP), 95% to ceftazidime (CAZ), tetracycline (TET) and sulfamethoxazole-trimethoprim (SXT), 30% to FOX and chloramphenicol (CHL), 85% to ciprofloxacin (CIP), enrofloxacin (ENR) and streptomycin (STR), and 65% to kanamycin (KAN). All the isolates were susceptible to meropenem (MEM). Among the 20 isolates, 1 (5%) was resistant to 2 classes of antibacterial agents while 19 (95%), including all the FOX-resistant strains, were resistant to ≥ 3 classes of antibacterial agents. The isolates exhibited 11 multiple antibacterial resistance patterns with AMP, CAZ, FOX, TET, CIP, ENR, STR, KAN, SXT being predominant. Conclusion: Healthy asymptomatic students in UNN are potential reservoirs and disseminators of ESC- and cephamycin (FOX)-resistant E. coli.

Beta-lactam antibiotics are one of the most important ones in clinical practice and most commonly prescribed drugs. There are few possible mechanisms of beta-lactam resistance. Beta-lactamase opens the b-lactam ring, deactivating the molecule's antibacterial properties, and among them, Extended-spectrum beta-lactamases (ESBL) are the most important. Antibiotic resistance gene can both be located on chromosome, or on plasmids. Betalactamases share the ability to hydrolyse penicillin, cephalosporin and monobactam, but not carbapenems and cephamycin, and yet, they are inhibited by clavulanic acid and/or tazobactam. Far most important in clinical practice are CTX-M enzymes, followed by TEM and SHV. Other less widespread ones are VEB, PER, GES, TLE, IBC, BES, BEL and others. Over the last years the presence of antibiotic resistant bacteria has been shown outside the clinical environment, including water, soil and food producing animals. If this problem persists in all over the world, carbapenems might be the first line antibiotics. Considering the present situation, antimicrobial resistance could be one of the greatest challenges in modern medicine, and it is important to ensure that antibiotics are prescribed only when needed.

Background: Streptomyces clavuligerus is prolific producer of cephamycin C, a medically important antibiotic. In our former study, cephamycin C titer was 2-fold improved by disrupting homoserine dehydrogenase (hom) gene of aspartate pahway in Streptomyces clavuligerus NRRL 3585. Objective: In this article, we aimed to provide a comprehensive understanding at the proteome level on potential complex metabolic changes as a consequence of hom disruption in Streptomyces clavuligerus AK39. Methods: A comparative proteomics study was carried out between the wild type and its hom disrupted AK39 strain by 2 Dimensional Electrophoresis-Matrix Assisted Laser Desorption and Ionization Time-Of-Flight Mass Spectrometry (2DE MALDI-TOF/MS) and Nanoscale Liquid Chromatography- Tandem Mass Spectrometry (nanoLC-MS/MS) analyses. Clusters of Orthologous Groups (COG) database was used to determine the functional categories of the proteins. The theoretical pI and Mw values of the proteins were calculated using Expasy pI/Mw tool. Results: “Hypothetical/Unknown” and “Secondary Metabolism” were the most prominent categories of the differentially expressed proteins. Upto 8.7-fold increased level of the positive regulator CcaR was a key finding since CcaR was shown to bind to cefF promoter thereby direcly controlling its expression. Consistently, CeaS2, the first enzyme of CA biosynthetic pathway, was 3.3- fold elevated. There were also many underrepresented proteins associated with the biosynthesis of several Non-Ribosomal Peptide Synthases (NRPSs), clavams, hybrid NRPS/Polyketide synthases (PKSs) and tunicamycin. The most conspicuously underrepresented protein of amino acid metabolism was 4-Hydroxyphenylpyruvate dioxygenase (HppD) acting in tyrosine catabolism. The levels of a Two Component System (TCS) response regulator containing a CheY-like receiver domain and an HTH DNA-binding domain as well as DNA-binding protein HU were elevated while a TetR-family transcriptional regulator was underexpressed. Conclusion: The results obtained herein will aid in finding out new targets for further improvement of cephamycin C production in Streptomyces clavuligerus.

Antimicrobial resistance (AMR) is a global threat gaining more and more practical significance every year. The protection of bacteria against antimicrobials based on antimicrobial resistance genes (ARGs) developed in evolution. One of the essential clinical questions is the origin of ARGs of pathogen bacteria. Since the bacteria can share genetic components by horizontal gene transfer (HGT), all even non-pathogen bacteria may provide ARG to any pathogens when they became close physically. The bacteria of the human gut may make contact with bacteria entered into the body by food. The fermented food contains bacteria in high amount by its nature. Here we studied the diversity of ARG content by a unified metagenomic approach in various kefir and yoghurt products, in grain and isolated bacterial strains. We found numerous ARGs of commonly used fermenting bacteria with diversity characteristics in kefir and yoghurt samples. Even with the strictest filter restrictions we identified ARGs undermining the efficacy of aminocoumarin, aminoglycoside, carbapenem, cephalosporin, cephamycin, diaminopyrimidine, elfamycin, fluoroquinolone, fosfomycin, glycylcycline, lincosamides, macrolide, monobactam, nitrofuran, nitroimidazole, penam, penem, peptide, phenicol, rifamycin, tetracycline and triclosan. In the case of genelmrD, we detected genetic environment providing mobility of this ARG. Our findings support that theory during the fermentation process the food ARG content can grow by the bacteria multiplication. Results presented suggest that starting culture strains of fermented food should be monitored and selected to decrease the ARG amount intake by nutrition.

Citrobacter freundii AMC0703 was isolated from the intestinal mucosa of an 11-year-old organ donor. Genome analysis revealed the presence of multiple factors potentially aiding in pathogenicity, including fimbriae, flagella, and genes encoding resistance to fluoroquinolones, cephamycin, fosfomycin, and aminocoumarin.

β-Lactams are the most important class of antibiotics, for which the emergence of resistance threatens their utility. As such, we explored the extent to which the tetramic acid motif, frequently found in naturally occurring antibiotics, can be used to generate novel β-lactam antibiotics with improved antibacterial activity. We synthesized new ampicillin – tetramic acid, cephalosporin – tetramic acid, and cephamycin – tetramic acid analogs and evaluated their activities against problematic Gram-positive and Gram-negative pathogens. Amongst the analogs, a 7-aminocephalosporanic acid analog, 3397, and a 7-amino-3-vinyl cephalosporanic acid, 3436, showed potent activities against S. aureus NRS 70 (MRSA) with MICs of 6.25 μg/mL and 3.13 μg/mL respectively. These new analogs were ≥16-fold more potent than cefaclor and cephalexin. Additionally, a Δ2 cephamycin – tetramic acid analog 3474 which contained a basic guanidinium substituent at the 5-position of the tetramic acid core displayed potent activity against several clinical strains of K. pneumoniae and E. coli.

Objective Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to public and clinical health because of their high levels of resistance to various antibiotics. We assessed the efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) against Imipenemase (IMP)-producing CRE, using the checkerboard method and time-killing assay on 13 Enterobacteriaceae isolates harboring blaIMP-1 (4 Enterobacter hormaechei, 5 Escherichia coli, and 4 Klebsiella pneumoniae isolates) and 13 isolates harboring blaIMP-6 (8 E. coli and 5 K. pneumoniae isolates). Results Minimum inhibitory concentrations (MICs) of MEM and CMZ ranged from 2 to 64 and 64 to 2048 μg/mL, respectively. Checkerboard method demonstrated the synergy of the MEM/CMZ combination in all the tested IMP-producing CRE isolates, and the time-kill assay indicated a bactericidal effect for both blaIMP-1 and blaIMP-6 positive CRE when MEM/CMZ combination was used. In vitro, the MEM/CMZ combination was potentially effective against IMP-1- or IMP-6-producing CRE. Further investigations including in vivo animal studies and clinical studies are warranted to corroborate the clinical utility of the novel combination therapy.

Many prophylactic antibiotic medications for use during transrectal prostate biopsy (TPBX) have been reported, but most reports did not classify the patients' risk of infection. Since fluoroquinolo...

Cefoxitin is a second-generation cephamycin antibiotic, which at concentrations ≥100 µg/mL is known to modestly interfere, for up to 2 hours post-infusion, with serum creatinine measurement via the traditional Jaffe-based assay. We report a case of a severe serum creatinine elevation while utilizing cefoxitin as a component of an antimicrobial regimen in a critically ill patient with Mycobacterium abscessus ventriculomeningitis. Our results, both via patient serum analysis and a cefoxitin spiking experiment, demonstrate interference despite the utilization of improved modern Jaffe-based assays. In fact, the cefoxitin-creatinine interference may be clinically relevant at concentrations 3 times lower than that listed in the package insert and may display more than a modest interference at typical therapeutic concentrations.

Streptomyces clavuligerus is a Gram‐positive bacterium that is a high producer of secondary metabolites with industrial applications. The production of antibiotics such as clavulanic acid or cephamycin has been extensively studied in this species; nevertheless, other aspects, such as evolution or ecology, have received less attention. Furthermore, genes that arise from ancient events of lateral transfer have been demonstrated to be implicated in important functions of host species. This approximation discovered relevant genes that genomic analyses overlooked. Thus, we studied the impact of horizontal gene transfer in the S. clavuligerus genome. To perform this task, we applied whole‐genome analysis to identify a laterally transferred sequence from different domains. The most relevant result was a putative antimicrobial peptide (AMP) with a clear origin in the Hymenoptera order of insects. Next, we determined that two copies of these genes were present in the megaplasmid pSCL4 but absent in the S. clavuligerus ATCC 27064 chromosome. Additionally, we found that these sequences were exclusive to the ATCC 27064 strain (and so were not present in any other bacteria) and we also verified the expression of the genes using RNAseq data. Next, we used several AMP predictors to validate the original annotation extracted from Hymenoptera sequences and explored the possibility that these proteins had post‐translational modifications using peptidase cleavage prediction. We suggest that Hymenoptera AMP‐like proteins of S. clavuligerus ATCC 27064 may be useful for both species adaptation and as an antimicrobial molecule with industrial applications.

The prevalence of multidrug-resistant gram-negative bacteria has continuously increased over the past few years; bacterial strains producing AmpC β-lactamases and/or extended-spectrum β-lactamases (ESBLs) are of particular concern. We combined high-resolution whole genome sequencing and phenotypic data to elucidate the mechanisms of resistance to cephamycin and β-lactamase in Korean Klebsiella pneumoniae strains, in which no AmpC-encoding genes were detected by PCR. We identified several genes that alone or in combination can potentially explain the resistance phenotype. We showed that different mechanisms could explain the resistance phenotype, emphasizing the limitations of the PCR and the importance of distinguishing closely-related gene variants.

Summary Streptomyces clavuligerus ATCC 27064 and S. clavuligerus ΔccaR::tsr cultures were grown in asparagine-starch medium, and samples were taken in the exponential and stationary growth phases. Transcriptomic analysis showed that the expression of 186 genes was altered in the ccaR-deleted mutant. These genes belong to the cephamycin C gene cluster, clavulanic acid gene cluster, clavams, holomycin, differentiation, carbon, nitrogen, amino acids or phosphate metabolism and energy production. All the clavulanic acid biosynthesis genes showed Mc values in the order of −4.23. The blip gene-encoding a β-lactamase inhibitory protein was also controlled by the cephamycin C-clavulanic acid cluster regulator (Mc −2.54). The expression of the cephamycin C biosynthesis genes was greatly reduced in the mutant (Mc values up to −7.1), while the genes involved in putative β-lactam resistance were less affected (Mc average −0.88). Genes for holomycin biosynthesis were upregulated. In addition, the lack of clavulanic acid and cephamycin production negatively affected the expression of genes for the clavulanic acid precursor arginine and of miscellaneous genes involved in nitrogen metabolism (amtB, glnB, glnA3, glnA2, glnA1). The transcriptomic results were validated by quantative reverse transcription polymerase chain reaction and luciferase assay of luxAB-coupled promoters. Transcriptomic analysis of the homologous genes of S. coelicolor validated the results obtained for S. clavuligerus primary metabolism genes.

Summary Objectives Urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are on the increase. Although cefmetazole is stable in vitro against the hydrolyzing activity of ESBLs, no clinical study has ever evaluated its role in infections caused by these organisms. We therefore evaluated the efficacy of cefmetazole compared to carbapenems against pyelonephritis caused by ESBL-producing Enterobacteriaceae . Methods A retrospective chart review was conducted at a tertiary care hospital from August 2008 to July 2010. Chart reviews were done for patients with ESBL-producing organisms in urine identified in the microbiology database. Patients who were treated with cefmetazole were compared to those treated with carbapenems. The clinical and bacteriological cure rates at 4 weeks after completion of therapy were evaluated. Results Two hundred and fifty-six urine cultures growing ESBL-producing organisms were identified during the study period. Ten patients treated with cefmetazole and 12 patients treated with carbapenems were evaluated. There was no difference in clinical (9/10 vs. 12/12, p =0.46) or bacteriological cure rate (5/7 vs. 6/7, p =1.00) at 4 weeks after the completion of therapy. There was no difference in the incidence of adverse effects (2/10 vs. 2/12, p =1.00). Conclusions Cefmetazole may be a useful option for the treatment of UTIs caused by ESBL-producing organisms. Prospective and larger sized studies are needed to confirm our findings.

One of the most common way that bacteria become resistant to antibiotics is by the production of metallo-β-lactamases (MβLs) to hydrolyze the β-lactam-containing antibiotics. In this paper, the thermodynamic parameters of benzylpenicillin hydrolysis with B1 subclasses MβL CcrA (carbapenem and cephamycin resistance) from Bacteroides fragilis were determined by microcalorimetry. The activation free energy Δ G ≠ θ is 87.90 ± 0.03, 88.99 ± 0.01, 89.93 ± 0.04 and 90.93 ± 0.05 kJ mol−1 at 293.15, 298.15, 303.15 and 308.15 K, activation enthalpy Δ H ≠ θ is 29.21 ± 0.03 kJ mol−1, activation entropy Δ S ≠ θ is −200.34 ± 0.08 J mol−1 K−1, the reaction order is 1.4, and the apparent activation energy E is 31.71 kJ mol−1. The thermodynamic characterization indicated that CcrA prefer to hydrolyze penicillins among three kinds of β-lactam-containing antibiotics.

Background: Infections due to extended spectrum β-lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae have become an important clinical problem. These organisms are important regarding the infection control by the physicians. Objective: The present study was undertaken to determine the prevalence of ESBLs along with their antimicrobial sensitivity pattern in Escherichia coli and Klebsiella pneumoniae . Methodology: This cross sectional study was conducted in the Department of Microbiology at Sir Salimullah Medical College, Dhaka. Urine samples were collected from patients who were clinically suspected to have UTI. After incubation, plates were checked for presence of suspected pathogens. Organisms were identified to species level by conventional methods. All isolated E. coli and K. pneumoniae were included in the study. The susceptibility to antibiotics was determined by Kirby Bauer method on Muller Hinton agar. Isolates were screened for ESBL production by using disk diffusion of cefotaxime, ceftazidime, ceftriaxone and cefpodoxime placed on inoculated plates containing Muller Hinton agar according to the CLSI recommendations. Phenotypic confirmatory test for ESBL producers was done by combined disc diffusion for all the isolates that were screened positive for the ESBL production following CLSI guidelines. Combined disk diffusion method was also done in this study. Result: A total of 220 non repeated urine samples were cultured of which 132(60%) cases had shown the bacterial growth. Among the 132 samples Escherichia coli had found in 103(78.0%) cases and Klebsiella spp. was found in 14(10.6%) cases. Out of 103 E coli 23(22.3%) cases was found as ESBL strain. On the other hand within 14 Klebsiella species, the ESBL strain was found in 5(35.7%) cases. Both E coli and Klebsiella species were 100% sensitive to imipenem. However, cephamycin was sensitive in 93.7% and 100% in E coli and Klebsiella species respectively. Conclusion: Results indicate that routine ESBL detection should be made imperative and empirical use of third generation cephalosporins must be discouraged. DOI: http://dx.doi.org/10.3329/jssmc.v4i1.11999 J Shaheed Suhrawardy Med Coll, 2012;4(1):22-25

RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

AmpC β-lactamases (Bla(AmpC)) are an emerging group of antimicrobial resistance determinants. The lack of an agreed Bla(AmpC) detection method hinders investigation of their epidemiology and understanding of their clinical significance. This study compared the sensitivity and specificity of phenotypic methods of Bla(AmpC) detection in a collection of 246 Enterobacteriaceae with a diverse range of β-lactam resistance profiles. The Bla(AmpC) screening methods evaluated were based on cephamycin, ceftazidime and cefepime susceptibility. These were compared with Bla(AmpC) screening using conventional ESBL detection methods. The confirmatory methods evaluated were biologically based assays, inhibitor-based assays, an AmpC Etest and a rapid chromogenic assay. A multiplex nucleic acid amplification test and the three-dimensional enzyme extraction assay were used as reference methods. Bla(AmpC) activity was present in 74 isolates. The majority of the enzymes were plasmid-encoded and belonged to the CMY, DHA and EBC families. The screening methods had sensitivities between 47 and 99 % and specificities of 45-95 %. The performance of confirmatory tests varied widely, ranging in sensitivity from 19 % to 97 % and in specificity from 88 % to 100 %. Only the Tris-EDTA and MAST ID D68C disc tests had a sensitivity and a specificity above 90 %. Further investigation is needed to establish the most suitable enzyme substrates, inhibitor types, inhibitor concentrations and interpretative cut-offs in order to refine the inhibitor-based methods. A simple disc-based protocol using cefoxitin non-susceptibility as a screening tool, followed by the Tris-EDTA method for confirmation, detects Bla(AmpC) activity with 95 % sensitivity and 98 % specificity.

The β-lactam classes of antibacterials are preeminent in the treatment of bacterial infection due to their unparalleled clinical efficacy and clinical safety. Following the discovery of the penicillins, successive β-lactam drug discovery has added the cephalosporin, penem cephamycin, clavulanate, monobactam, nocardicin, and carbapenem subclasses. The driving force behind much of this era of discovery is the staggering ability of pathogenic bacteria to adapt previous generations of the β-lactam by the acquisition and expression of resistance mechanisms. Although many factors contribute to β-lactam resistance, alterations to the molecular targets of the β-lactams (the penicillin binding proteins) and the use of enzymes (the β-lactamases) capable of the hydrolytic deactivation of the β-lactams are paramount. This review traces the historical development of β-lactam drug discovery, with emphasis on the most recent progress in the medicinal chemistry, biochemistry, and microbiology of the β-lactams leading to the discovery of new generation β-lactam antibacterials effective against the Gram-negative and -positive bacterial pathogens of current medical concern. Keywords: β-lactam; β-lactamase; penicillin; cephalosporin; monobactam

It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.

Three open reading frames denoted as orf21, orf22, and orf23 were identified from downstream of the currently recognized gene cluster for clavulanic acid biosynthesis in Streptomyces clavuligerus ATCC 27064. The new orfs were annotated after in silico analysis as genes encoding a putative sigma factor, a sensor kinase, and a response regulator. The roles of the individual genes were explored by disruption of the corresponding orfs, and the morphological and antibiotic production phenotypes of the resulting mutants were compared. In orf21 and orf22 mutants, no growth or morphological differences were noted, but modest reduction of cephamycin C (orf21), or both cephamycin C and clavulanic acid production (orf22) compared with wild-type, were observed. In orf23 mutant, cell growth and sporulation was retarded, and clavulanic acid and cephamycin C production were reduced to 40 and 47% of wild-type levels, respectively. Conversely, overexpression of orf23 caused precocious hyperproduction of spores on solid medium, and antibiotic production was increased above the levels seen in plasmid control cultures. Transcriptional analyses were also carried out on orf23 and showed that mutation had little effect on transcription of genes associated with the early stages of cephamycin C or clavulanic acid production but transcription of claR, which regulates the late stages of clavulanic acid production, was reduced in orf23 mutants. These observations suggest that the orf23 product may enable S. clavuligerus to respond to environmental changes by altering cell growth and differentiation. In addition, the effects of ORF23 on growth might indirectly regulate the biosynthesis of secondary metabolites such as clavulanic acid and cephamycin C.

Biosynthesis of cephamycin C in Streptomyces clavuligerus involves the initial conversion of lysine to α-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the lat gene causes complete loss of cephamycin production, pcd mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by pcd mutants could be restored to wild-type levels either by supplementation of the growth medium with α-aminoadipic acid or by complementation of the mutation with an intact copy of the pcd gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second pcd gene. Furthermore, cell extracts from pcd mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that S. clavuligerus must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis.

Forty-six cephamycin-resistant Escherichia coli isolates from patients diagnosed with community-acquired urinary tract infection were selected in order to study their resistance mechanism. With the exception of one isolate producing CMY-4, all isolates produced a CMY-2 β-lactamase. Molecular typing showed that the CMY-2-producing isolates were not related. Cephamycin resistance was plasmid encoded and conjugatively transferred. Plasmid digest profiles suggested that the plasmids were different. Thirty-nine of the 45 CMY-2-producing isolates harboured a plasmid containing a specific DNA fragment, IS Ecp1 – bla CMY-2 – blc – sugE , which was identical to those previously published in CMY-2-producing Salmonella enterica serotype Choleraesuis (SCB67) and Salmonella enterica serotype Typhimurium (pNF1358) from Taiwan and the USA, respectively. Among the remaining six isolates, insertion of IS 1294 and IS 1 at different positions was observed in one and five isolates, respectively. The regions surrounding bla CMY-2 of the six isolates were identical to the other 39 isolates as well as to SCB67 and pNF1358. Since the present identical transmissible bla CMY-2 -carrying element was observed in food animal sources both in the USA and Taiwan, its possible transmission to humans, as revealed in this study, is of great concern. Awareness of this mobile resistance element is required to prevent introduction into hospitals and to reduce the spread of this emerging resistance within the community.

To investigate the in vitro activities of cephamycins (cefmetazole and flomoxef) for extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC β-lactamase (pAmpC)-producing Enterobacteriaceae, a total of 574 third-generation cephalosporin-resistant clinical isolates were collected at a Japanese multicenter study. PCR and sequencing identified 394 isolates with only ESBL genes, 63 isolates with only pAmpC genes, and 6 isolates with both ESBL and pAmpC genes. blaCTX-M types predominated 95.5% of the ESBL genes, and blaCMY-2 predominated 91.3% of the pAmpC genes. The MIC50/90 values of cefmetazole and flomoxef were ≤ 1/4 and ≤ 1/≤ 1 μg/mL for isolates with only ESBL genes, respectively, and 16/>16 and 8/16 μg/mL for isolates with only pAmpC genes, respectively. Flomoxef ≥ 4 μg/mL had the best screening performance for the detection of isolates with pAmpC genes. Flomoxef had better in vitro activities against ESBL-producing Enterobacteriaceae and provided a clearer distinction between ESBL and pAmpC-producing Enterobacteriaceae compared to cefmetazole.

Central steps in the biosynthetic pathways of some of the most commonly used antibiotics, the cephalosporins, are catalysed by 2-oxoglutarate (2OG)-dependent oxygenases. Deacetoxycephalosporin C synthase (DAOCS) catalyses the 2OG-dependent oxidative expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydrothiazine ring of the cephalosporin nucleus. DAOCS uses dioxygen to create a reactive iron–oxygen intermediate from ferrous ion to drive the reaction. In prokaryotic cephalosporin producers, the cephalosporin product, DAOC, is hydroxylated at the 3′-position to form deacetylcephalosporin C (DAC) as catalysed by a second 2OG-dependent enzyme, DAC synthase (DACS). In eukaryotic cephalosporin producers, the reaction is catalysed by a bifunctional enzyme, DAOC/DACS, that catalyses both the ring expansion and the 3′-hydroxylation reactions. The prokaryotic and eukaryotic enzymes are closely related to DAOCS by sequence, suggesting these enzymes may have evolved by gene duplication. Cephamycin C-producing microorganisms use two enzymes, encoded by the genes cmcI/J, to convert cephalosporins to their 7α-methoxy derivatives that are less vulnerable to β-lactam hydrolysing enzymes. The methoxylation reaction is dependent on Fe(ii), 2OG and S-adenosylmethionine, suggesting the involvement of another 2OG-dependent oxygenase. Herein, structural and mechanistic features are summarized for these 2OG enzymes that utilize this common and flexible mode of dioxygen activation.

In 2013, an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 occurred in a nursery school in Japan. The outbreak affected 12 school children and five members of their families. All 17 isolates obtained from these individuals were found to be clonal, as determined by pulsed-field gel electrophoresis analysis and multilocus variable number tandem repeat analysis. The antimicrobial susceptibility profiles of the isolates to 20 drugs were examined, with three isolates showing resistance to the extended-spectrum cephalosporins (ESC) and cephamycin, including cefotaxime, ceftazidime, and cefminox. The resistant isolates carried the blaCMY-2 AmpC β-lactamase gene. It is proposed that the ESC-resistant EHEC O157:H7 isolates might have acquired the resistance plasmid encoding the blaCMY-2 gene during human to human infection in the nursery school.

The transcriptional and translational control of the biosynthesis of the beta-lactamase inhibitor clavulanic acid is a subject of great scientific and industrial interest. To study the role of the ribosomal protein L11 on control of clavulanic acid gene transcription, the DNA region aspC-tRNA(trp)-secE-rplK-rplA-rplJ-rplL of Streptomyces clavuligerus was cloned and characterized. An S. clavuligerus rplK(DeltaPALG) mutant, with an internal 12 nucleotides in-frame deletion in the rplK gene, encoding the L11 (RplK) ribosomal protein lacking amino acids (29)PALG(32), was constructed by gene replacement. This deletion alters the L11 N-terminal domain that interacts with the RelA and class I releasing factors-mediated translational termination. The mutant grew well, showed threefold higher resistance to thiostrepton, did not form spores and lacked diffusible brown pigments, as compared with the wild-type strain. The wild-type phenotype was recovered by complementation with the native rplK gene. S. clavuligerus rplK(DeltaPALG) produced reduced levels of clavulanic acid (15-26% as compared with the wild type) and cephamycin C (40-50%) in cultures grown in defined SA and complex TSB media. The decreased yields resulted from an impaired transcription of the regulatory genes ccaR and claR and the cefD and ceaS2 genes for cephamycin and clavulanic acid biosynthesis respectively. Expression of ceaS2 encoding carboxyethylarginine synthase (CEAS), the precursor-committing enzyme for clavulanic acid biosynthesis, was particularly affected in this mutant. In the wild-type strain polyphosphorylated nucleotides peaked at 36-48 h of growth in SA cultures whereas expression of the cephamycin and clavulanic acid genes occurred 12-24 h earlier than the increase in ppGpp indicating that there is no strict correlation between the peak of ppGpp and the onset of transcription of the clavulanic acid and cephamycin C biosynthesis. The drastic effect of the rplK(DeltaPALG) mutation on the onset of expression of the ceaS2 and the regulatory ccaR and claR genes and the lack of correlation with ppGpp levels suggest that the onset of transcription of these genes is modulated by the conformational alteration of the N-terminal region of L11 probably by interaction with the nascent peptide releasing factors and with RelA.

Many secondary metabolites (e.g. antibiotics and mycotoxins) are toxic to the microorganisms that produce them. The clusters of genes that are responsible for the biosynthesis of secondary metabolites frequently contain genes for resistance to these toxic metabolites, such as different types of multiple drug resistance systems, to avoid suicide of the producer strains. Recently there has been research into the efflux systems of secondary metabolites in bacteria and in filamentous fungi, such as the large number of ATP-binding cassette transporters found in antibiotic-producing Streptomyces species and that are involved in penicillin secretion in Penicillium chrysogenum. A different group of efflux systems, the major facilitator superfamily exporters, occur very frequently in a variety of bacteria that produce pigments or antibiotics (e.g. the cephamycin and thienamycin producers) and in filamentous fungi that produce mycotoxins. Such efflux systems include the CefT exporters that mediate cephalosporin secretion in Acremonium chrysogenum. The evolutionary origin of these efflux systems and their relationship with current resistance determinants in pathogenic bacteria has been analyzed. Genetic improvement of the secretion systems of secondary metabolites in the producer strain has important industrial applications.

On the basis of molecular dynamics simulations, we investigate the dynamic properties of the carbapenem- and cephamycin-resistant dinuclear zinc metallo-beta-lactamase from Bacteroides fragilis and its complex with a biphenyl tetrazole inhibitor, 2-butyl-6-hydroxy-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-quinazolin-4-one 1 (L-159061). The results obtained with the newly developed force field parameters for the coordination environment of the catalytic zinc ions show that the active site gorge comprising major and minor loops gets deeper and narrower upon binding of the inhibitor, which supports the previous experimental implication that the structural flexibility of the loop structures plays a significant role in enzymatic action. In the presence of the inhibitor, the Trp32 side chain at the apex of the major loop covers the entrance of active site channel, thereby contributing to the stabilization of the enzyme-inhibitor complex. In addition to a direct coordination of the inhibitor tetrazole ring to the second zinc ion in the active site, the hydrogen bonding of Lys167 to the inhibitor carbonyl group and hydrophobic interactions between the inhibitor and side chains of loop residues prove to be significant binding forces of the enzyme-inhibitor complex.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.

The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 12·1 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed: ORF10–11, ORF12–13 and ORF15–16–17–18. Potential transcriptional terminators were identified downstream of ORF11 (fd) and ORF15. Targeted disruption of ORF10 (cyp) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10–14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid.

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149 H240 , and TEM-149 H164-H240 versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149 H240 , and TEM-149 H164-H240 β-lactamases, and the K i (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k +2 and k +3 (first-order acylation and deacylation constants, respectively), and k +2 / K values were calculated.

Many early studies on penicillin and cephamycin biosynthesis were carried out in Streptomyces clavuligerus, but because of the commercial importance of clavulanic acid, most recent studies have focused on that compound. In particular, genetic engineering to generate improved clavulanic acid producer strains has been a main interest. S. clavuligerus grows and produces secondary metabolites optimally at 28 to 30°C, but it is unusually sensitive to elevated temperature and will not grow above 31 to 32°C. Liquid media employed by research groups studying the production of the various types of β-lactam metabolites produced by S. clavuligerus vary widely. Numerous bioprocess-type studies examining growth medium optimization as a means of improving production of clavulanic acid have appeared in recent years, typically reinforcing the association of soy-based growth medium constituents with high productivity. In S. clavuligerus, biosynthesis begins with the amino acids L-lysine, L-cysteine, and L-valine and proceeds through the synthesis of penicillin and cephalosporin intermediates to give the final cephamycin product. While improvements in productivity can be achieved by increasing production of pathway enzymes, another approach to achieving this is to identify genes encoding critical regulatory proteins and increase their expression levels. More global approaches to improvement of antibiotic production could also be coupled with strategies aimed at the pathway-specific regulators. Effects of greater magnitude are seen when pathway-specific regulators are targeted for overproduction, and combination approaches targeting both global and specific regulators along with individual pathway enzymes for overproduction, together with elimination of competing pathways, will likely yield even greater results.

The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC-MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains.

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.

cysteine is a precursor of the penicillin, cephalosporin and cephamycin families of β-lactam antibiotics. Cystathionine-γ-lyase (encoded by the mecB gene), an enzyme that splits cystathionine releasing cysteine, is required for high-level cephalosporin production in methionine-supplemented medium. By amplification of the mecB gene in Acremonium chrysogenum C10, several transformants were obtained that produced 10-40% higher levels of cephalosporin. All selected transformants contained at least two or three copies of the mecB gene as shown by Southern hybridization with a probe internal to mecB. Two of these transformants, A. chrysogenum T27 and A. chrysogenum T58, showed 4- to 10-fold higher cystathionine-γ-lyase activity than the control strain. Northern hybridizations indicated that the levels of the two mecB transcripts of 1.7 and 1.5 kb were greatly increased in transformants T27 and T58. Fermentor studies using controlled conditions confirmed that transformant T27 was a cephalosporin overproducer, reaching titers of nearly 2000 μg/ml of cephalosporin in Shen-defined medium that correlated with two- to fourfold higher cystathionine-γ-lyase levels than in the control strain. Transformant T58 containing five- to sixfold higher levels of cystathionine-γ-lyase in fermentor cultures showed a reduced growth rate and a slow cephalosporin accumulation rate. In conclusion, moderately increased levels of cystathionine-γ-lyase stimulated cephalosporin production but very high levels of this enzyme were deleterious for growth and cephalosporin biosynthesis. Journal of Industrial Microbiology & Biotechnology (2001) 27, 252–258.

The first two genes of the lysine pathway are closely linked forming a transcriptional operon in the cephamycin producer ‘Amycolatopsis lactamdurans’. The asd gene, encoding the enzyme aspartic semialdehyde dehydrogenase, has been cloned by complementation of Escherichia coli asd mutants. It encodes a protein of 355 aa with a deduced M r of 37109. The ask gene encoding the aspartokinase (Ask) is located upstream of the asd gene as shown by determination of Ask activity conferred to E. coli transformants. asd and ask are separated by 2 nt and are transcribed in a bicistronic 2·6 kb mRNA. As occurs in corynebacteria, the presence of a ribosome-binding site within the ask sequence suggests that this ORF encodes two overlapping proteins, Askα of 421 aa and M r 44108, and Askβ of 172 aa and M r 18145. The formation of both subunits of Ask from a single gene (ask) was confirmed by using antibodies against the C-terminal end of Ask which is identical in both subunits. Ask activity of ‘A. lactamdurans’ is regulated by the concerted action of lysine plus threonine and this inhibition is abolished in E. coli transformants containing Ser301 to Tyr, or Gly345 to Asp mutations of the ‘A. lactamdurans’ ask gene.

Two protein bands with strong esterase activity are present in broths of Nocardia lactamidurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed Km values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C.

Summary The cephamycin producer strain ‘ Nocardia lactamdurans ’ contains four ribosomal RNA ( rrn ) operons. One of them ( rrn D) was cloned from a DNA library in the bifunctional cosmid pJAR4. A 2229 bp region of rrn D has been sequenced. The ‘ N. lactamdurans ’ rrn D operon maintains the canonical order 5′-16S-23S-5S-3′. Four of the consensus Gurtler-Stanisch sequences were found in the 16S rRNA gene and a fifth one in the sequenced 5′ region of the 23S rRNA gene. The and Shine-Dalgarno sequence of ‘ N. lactamdurans ’ (located in the 3′-end of the 16S rRNA gene) was found to be 5′-CCUCCUUUCU-3′ and is identical to that of Corynebacterium lactofermentum and Mycobacterium tuberculosis . A phylogenetic analysis of ‘ N. lactamdurans ’ by the neighbor-joining method using the entire 16S rRNA nucleotide sequence revealed that this actinomycete is closely related to Amycolatopsis orientalis subsp orientalis, Amycolatopsis coloradensis, Amycolatopsis alba, Amycolatopsis sulphurea and other Amycolatopsis sp. but only distantly related to species of the genus Nocardia . The cephamycin producer ‘ N. lactamdurans ’ NRRL 3802 should be, therefore, classified as Amycolatopsis lactamdurans . The deduced secondary structure of the 16S rRNA is very similar to that of A. colorandensis and A. alba but different from those of species of the Nocardia genus supporting the incorporation of ‘ N. lactamdurans ’ into the genus Amycolatopsis .

In vitro synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus was investigated using alpha-ketoglutarate, L-ascorbic acid, FeSO(4), S-adenosyl-L-methionine, and 7alpha-demethoxycefminox as the substrates. The formation of cefminox was detected both by a biological assay with Proteus vulgaris GN 76/C-1 and by high performance liquid chromatography. Although the conversion rate of 7alpha-demethoxycefminox to cefminox was observed to be quite low, it still demonstrated the potential for an enzymatic process to replace the chemical steps which are currently in use for the production of cefminox.

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans , in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbAB promoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans . Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

The discovery and development of the β-lactam antibiotics are among the most powerful and successful achievements of modern science and technology. Since Fleming's accidental discovery of the penicillin-producing mold, seventy years of steady progress has followed, and today the β-lactam group of compounds are the most successful example of natural product application and chemotherapy. Following on the heels of penicillin production by Penicillium chrysogenum came the discoveries of cephalosporin formation by Cephalosporium acremonium, cephamycin, clavam and carbapenem production by actinomycetes, and monocyclic β-lactam production by actinomycetes and unicellular bacteria. Each one of these groups has yielded medically-useful products and has contributed to the reduction of pain and suffering of people throughout the world. Research on the microbiology, biochemistry, genetics and chemistry of these compounds have continued up to the present with major contributions being made by both individual and collaborative groups from industry and academia. The discovery of penicillin not only led to the era of the wonder drugs but provided the most important antibiotics available to medicine. Continued efforts have resulted in the improvement of these compounds with respect to potency, breadth of spectrum, activity against resistant pathogens, stability and pharmacokinetic properties. On the research front, major advances are being made on structural and regulatory biosynthetic genes and metabolic engineering of the pathways involved. New semisynthetic compounds especially those designed to combat resistance development are being examined in the clinic, and unusual non-antibiotic activities of these compounds are being pursued. Although seventy years of age, the β-lactams are not yet ready for retirement.

Cephamycin C is produced in a nine steps pathway by the actinomycetes S. clavuligerus and N. lactamdurans. The genes encoding the biosynthesis enzymes are clustered in both microorganisms as well as in the cephabacin producer Lysobacter lactamgenus, a Gram negative bacterium. The clusters of genes include genes encoding enzymes common to the biosynthesis of penicillin and cephalosporin C by the eukaryotic producers Penicillium chrysogenum and Cephalosporiun acremonium and genes for steps specific for the formation of the precursor α-aminoadipic acid as well as for the enzymes involved in the late modification of the cephalosporin intermediates of the pathway. Present are also genes for proteins involved in the export and/or resistance to cephamycin C. In S. clavuligerus a gene encoding a regulatory protein controlling the formation of cephamycin C and clavulanic acid is also present in the cluster.

Nutritional requirements of a mutant strain of Streptomyces clavuligerus for Cephamycin C production were derived by testing various indigenous substrates and their concentrations in production medium. Upon initial screening, six components: tapioca starch (TS), corn steep liquor (CSL), sunflower cake (SFC), K 2 HPO 4 , MgSO 4 .7H 2 O and MnSO 4 were identified as supporting high production of Cephamycin C. Statistical analysis (ANOVA) of media components indicated that addition of oil cake SFC to the medium enhanced production significantly. SFC controlled foam during fermentation and served as a pH regulator.

Batch cultures of Nocardia lactamdurans MA4213 in stirred tank fermentors (400 rpm) became oxygen limited after 45 h of incubation. When the stirrer speed was increased at 36 h from 400 to 600 rpm there was a 5-fold increase in the dissolved oxygen (DO) in the broth what resulted in a 2-fold stimulation of cephamycin biosynthesis. Immunoblotting studies using antibodies against the α-aminoadipyl-cysteinyl-valine synthetase and the protein P7 (cephem-7-hydroxylase) component of the methoxylation system showed that cultures with increased DO concentrations maintained high levels of these proteins for an extended period of time. Under these conditions there was an enhanced conversion of cephamycin intermediates into cephamycin C. Allophane and alumina exerted a strong stimulatory effect on cephamycin biosynthesis that correlated with high DO levels in allophane- or alumina-supplemented cultures at low agitation speeds. Allophane or alumina supplementation of N. lactamdurans cultures resulted in a delayed decay of the cephem-7-hydroxylase protein in the cells favoring conversion of the cephamycin intermediates into cephamycin C.

Delta-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into alpha-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 microM) and NAD+ (apparent Km 115 microM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6 amino-transferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but no in actinomycetes that do no produce beta-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to alpha-aminoadipic acid.

The ß-lactam antibiotics continue providing health to the world population by virtue of industrial production and discoveries of new molecules with useful activities. Sales of these remarkable compounds, including penicillins, cephalosporins, cefoxitin, monobactams, clavulanic acid, and carbapenems, have reached over $20 billion dollars per year. Strain improvement of the penicillin-producing strains of Penicillium chrysogenum has been truly remarkable, with present strains producing about 100,000 times more penicillin than the original Penicillium notatum of Sir Alexander Fleming. The traditional strain improvement programs based on random mutation and screening in combination with recombinant DNA techniques allowed an impressive ß-lactam yield enhancement at industrial scale. A remarkable amount of information has been gathered on the biosynthetic enzymes involved, the pathways of biosynthesis of ß-lactams as well as their regulation, and the genomics and proteomics of the producing organisms. The rational metabolic engineering of ß-lactam-producing microorganisms has resulted in productivity increments and the design of biosynthetic pathways giving rise to new antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms (GMOs). Modern aspects of the processes are discussed in the present review including genetics, molecular biology, metabolic engineering, genomics, and proteomics. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.