Penicillin-Binding Proteins, ß-Lactamases, and ß-Lactamase Inhibitors in ß-Lactam-Producing Actinobacteria: Self-Resistance Mechanisms.

The human society faces a serious problem due to the widespread resistance to antibiotics in clinical practice. Most antibiotic biosynthesis gene clusters in actinobacteria contain genes for intrinsic self-resistance to the produced antibiotics, and it has been proposed that the antibiotic resistance genes in pathogenic bacteria originated in antibiotic-producing microorganisms. The model actinobacteria Streptomyces clavuligerus produces the ß-lactam antibiotic cephamycin C, a class A ß-lactamase, and the ß lactamases inhibitor clavulanic acid, all of which are encoded in a gene supercluster; in addition, it synthesizes the ß-lactamase inhibitory protein BLIP. The secreted clavulanic acid has a synergistic effect with the cephamycin produced by the same strain in the fight against competing microorganisms in its natural habitat. High levels of resistance to cephamycin/cephalosporin in actinobacteria are due to the presence (in their ß-lactam clusters) of genes encoding PBPs which bind penicillins but not cephalosporins. We have revised the previously reported cephamycin C and clavulanic acid gene clusters and, in addition, we have searched for novel ß-lactam gene clusters in protein databases. Notably, in S. clavuligerus and Nocardia lactamdurans, the ß-lactamases are retained in the cell wall and do not affect the intracellular formation of isopenicillin N/penicillin N. The activity of the ß-lactamase in S. clavuligerus may be modulated by the ß-lactamase inhibitory protein BLIP at the cell-wall level. Analysis of the ß-lactam cluster in actinobacteria suggests that these clusters have been moved by horizontal gene transfer between different actinobacteria and have culminated in S. clavuligerus with the organization of an elaborated set of genes designed for fine tuning of antibiotic resistance and cell wall remodeling for the survival of this Streptomyces species. This article is focused specifically on the enigmatic connection between ß-lactam biosynthesis and ß-lactam resistance mechanisms in the producer actinobacteria.

The Use of Noncarbapenem ß-Lactams for the Treatment of Extended-Spectrum ß-Lactamase Infections.

The continued rise in infections caused by extended-spectrum ß-lactamase (ESBL)-producing pathogens is recognized globally as one of the most pressing concerns facing the healthcare community. Carbapenems are widely regarded as the antibiotics of choice for the treatment of ESBL-producing infections, even when in vitro activity to other ß-lactams has been demonstrated. However, indiscriminant carbapenem use is not without consequence, and carbapenem overuse has contributed to the emergence of carbapenem-resistant Enterobacteriaceae. The use of non-carbapenem ß-lactams for the treatment of ESBL infections has yielded conflicting results. In this review, we discuss the available data for the use of cephamycins, cefepime, piperacillin-tazobactam, ceftolozane-tazobactam, and ceftazidime-avibactam for the treatment of ESBL infections.

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission.

Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n = 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low. IMPORTANCE: ESBL-/pAmpC-producing enterobacteria occur frequently in broiler-fattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpC-positive broiler parent flocks as well as the hatchlings and the environment of the hatchery.

Evolutionary ecology of beta-lactam gene clusters in animals.

Beta-lactam biosynthesis was thought to occur only in fungi and bacteria, but we recently reported the presence of isopenicillin N synthase in a soil-dwelling animal, Folsomia candida. However, it has remained unclear whether this gene is part of a larger beta-lactam biosynthesis pathway and how widespread the occurrence of penicillin biosynthesis is among animals. Here, we analysed the distribution of beta-lactam biosynthesis genes throughout the animal kingdom and identified a beta-lactam gene cluster in the genome of F. candida (Collembola), consisting of isopenicillin N synthase (IPNS), δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS), and two cephamycin C genes (cmcI and cmcJ) on a genomic scaffold of 0.76 Mb. All genes are transcriptionally active and are inducible by stress (heat shock). A beta-lactam compound was detected in vivo using an ELISA beta-lactam assay. The gene cluster also contains an ABC transporter which is coregulated with IPNS and ACVS after heat shock. Furthermore, we show that different combinations of beta-lactam biosynthesis genes are present in over 60% of springtail families, but they are absent from genome- and transcript libraries of other animals including close relatives of springtails (Protura, Diplura and insects). The presence of beta-lactam genes is strongly correlated with an euedaphic (soil-living) lifestyle. Beta-lactam genes IPNS and ACVS each form a phylogenetic clade in between bacteria and fungi, while cmcI and cmcJ genes cluster within bacteria. This suggests a single horizontal gene transfer event most probably from a bacterial host, followed by differential loss in more recently evolving species.

The times they are a-changin': carbapenems for extended-spectrum-ß-lactamase-producing bacteria.

Several antimicrobial agents are being investigated as alternatives to carbapenems in the treatment of infections caused by ESBL-producing Enterobacteriaceae, which may be useful in avoiding overuse of carbapenems in the context of recent global spread of carbapenem-resistant Enterobacteriaceae. The most promising candidates for invasive infections so far are ß-lactam/ß-lactamase inhibitor combinations and cephamycins.

[Correlation analysis on combined medication with of Xiyanping injection in treatment of lung infection in real world].

To analyze the regularity in combined medication with Xiyanping injection (Xiyanping for short) in the real world by as- sociation rules. Totally 5 822 patients using Xiyanping injection was collected from the 18 Class III Grade I hospitals nationwide to study the combined medication information of the patient with lung infection and make the analysis by using association rules and Apriori. According to the results, major drugs combined with Xiyanping in treatment of lung infection included compound amino acid, inosine, coenzyme A, cytidine triphosphate, vitamin C. Common drugs combined with Xiyanping can be divided into 5 categories: nutrition support therapy (vitamin C, compound amino acid) , coenzymes (coenzyme A, cytidine triphosphate, inosine), expectorants and antiasthmatics (ambroxol, salbutamol, doxofylline), hormones (dexamethasone, budesonide), antibiotics (mainly cefminox). The main combined medicines mostly conformed to the regularity for drugs treating lung infection. In addition, there were two most common medical combination models: the model for Xiyanping combined a single medicine is Xiyanping + nutrition support therapy, while the model for Xiyanping combined two or more than two medicines is Xiyanping + nutrition support therapy + coenzyme. Pharmacologically, Xiyanping is mostly combined with western medicines with similar pharmacological effects to substitute or supplement the antibiotic effect in treating lung infection. However, further studies shall be conducted for the safety and rationality of the combined medication based on clinical practices, in order to provide reference for clinical medication.

Design, Synthesis, and Evaluation of Cephamycin-Based Antisporulation Agents targeting Clostridioides difficile.

With the aim of discovering small molecule inhibitors of the sporulation process in Clostridioides difficile, we prepared a series of C-7 α-(4-substituted-1H-1,2,3-triazol-1-yl)acetamide analogues of cefotetan, a known inhibitor of the C. difficile sporulation-specific protein target CdSpoVD. These analogues were evaluated using both in vitro binding assays with CdSpoVD and antisporulation assays against C. difficile. Further design concepts were aided utilizing the predicted docking scores (DS) using both AlphaFold (AF) models, and a crystal structure of the CdSpoVD protein (PDB 7RCZ). Despite being 1 order of magnitude more potent as a sporulation inhibitor than cefotetan, in vivo studies on compound 6a in a murine-model of C. difficile infection demonstrated comparable spore shedding capabilities as cefotetan. Importantly, compound 6a had no concerning broad spectrum antibacterial activities, toxicity, or hemolytic activity and thus has potential for further drug development.

Enhancing effect of lysine combined with other compounds on cephamycin C production in Streptomyces clavuligerus.

BACKGROUND: Lysine plays an important role in Streptomyces clavuligerus metabolism; it takes part in its catabolism, via cadaverine, and in its secondary metabolism, in which lysine is converted via 1-piperideine-6-carboxylate to alpha-aminoadipic acid, a beta-lactam antibiotic precursor. The role of lysine as an enhancer of cephamycin C production, when added to production medium at concentrations above 50 mmol l(-1), has already been reported in the literature, with some studies attributing a positive influence to multifunctional diamines, among other compounds. However, there is a lack of research on the combined effect of these compounds on antibiotic production. RESULTS: Results from experimental design-based tests were used to conduct response surface-based optimization studies in order to investigate the synergistic effect of combining lysine with cadaverine, putrescine, 1,3-diaminopropane, or alpha-aminoadipic acid on cephamycin C volumetric production. Lysine combined with cadaverine influenced production positively, but only at low lysine concentrations. On the whole, higher putrescine concentrations (0.4 g l(-1)) affected negatively cephamycin C volumetric production. In comparison to culture media containing only lysine as additive, combinations of this amino acid with alpha-aminoadipic acid or 1,3-diaminopropane increased cephamycin C production by more than 100%. CONCLUSION: This study demonstrated that different combinations of lysine with diamines or lysine with alpha-aminoadipic acid engender significant differences with respect to antibiotic volumetric production, with emphasis on the benefits observed for lysine combined with alpha-aminoadipic acid or 1,3-diaminopropane. This increase is explained by mathematical models and demonstrated by means of bioreactor cultivations. Moreover, it is consistent with the positive influence of these compounds on lysine conversion to alpha-aminoadipic acid, a limiting step in cephamycin C production.

Role of the cmcH-ccaR intergenic region and ccaR overexpression in cephamycin C biosynthesis in Streptomyces clavuligerus.

The effect of the CcaR regulatory protein on expression of the cephamycin C gene cluster is studied. Quantitative reverse transcription PCR (qRT-PCR) expression analysis of the cephamycin biosynthesis genes in the ccaR-disrupted strain, S. clavuligerus ccaR::aph, revealed that in the absence of CcaR, the lat and cmcI genes expression was reduced 2,200- and 1,087-fold compared with the wild type. Expression of pcbAB-pcbC-cefD-cefE-cmcJ-cmcH and blp was 225- to 359-fold lower, while expression of pcbR-pbpA-bla and orf10 was only slightly affected if at all, indicating that resistance and regulatory genes are not under CcaR control as opposed to pathway biosynthetic genes. In the intergenic cmcH-ccaR region, a small messenger RNA (mRNA) overlaps with the cmcH transcription terminator. Deletion of 688 bp of the intergenic region results in a strain, S. clavuligerus ΔRI, still able to produce cephamycin C and clavulanic acid but at levels 30-40% lower than the parental strain. Therefore, specific sequences in the intergenic region upstream of ccaR enhance the expression of ccaR but are not essential for its expression. Strains containing an additional ccaR gene integrated in the chromosome, S. clavuligerus pSET-PC, or multiple copies of ccaR expressed from the PglpF promoter, S. clavuligerus pAK23, were constructed. Fermentations of the pAK23 strain resulted in a 6.1-fold increase in specific cephamycin C production relative to the wild type. In the same experiments, qRT-PCR analysis of the cephamycin biosynthesis genes showed a 5.1-fold increase in ccaR expression and similar increases in expression of lat and cmcI, while expression of other cluster genes were increased in the order of 2- to 3-fold.

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism.

To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.

In vitro efficacy of cephamycins against multiple extended-spectrum ß-lactamase-producing Klebsiella pneumoniae, Proteus mirabilis, and Enterobacter cloacae isolates from dogs and cats.

The susceptibility of 218 extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae isolates from companion animals to three cephamycins (cefmetazole, flomoxef, and latamoxef) was investigated. Phenotypic testing found 8 of 120 Klebsiella pneumoniae (KP) and 15 of 69 Enterobacter cloacae (EC) isolates were ESBL and AmpC ß-lactamase (ABL) co-producers. Isolates of KP, Proteus mirabilis, and EC that only produced ESBL exhibited susceptibility rates to cefmetazole (95.5%, 82.7%, and 9.3%), flomoxef (99.1%, 96.6%, and 74.0%), and latamoxef (99.1%, 100%, and 100%), respectively. Notably, isolates of KP and EC co-producing ESBL and ABL had significantly lower susceptibility rates to the studied drugs when compared with only ESBL producers. This implies that the in vitro activity of cephamycins against ESBL-producing bacteria can differ depending on ABL production and bacterial species.

Characterization of DNA-binding sequences for CcaR in the cephamycin-clavulanic acid supercluster of Streptomyces clavuligerus.

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.

Molecular characterization of extended-spectrum beta-lactamases and its correlation with clinical laboratory standards institute interpretive criteria for disk diffusion susceptibility testing in enterobacteriaceae isolates in Thaialnd.

We performed extended-spectrum beta-lactamase (ESBL) phenotypic testing and molecular characterization of three ESBL genes (TEM, SHV and CTX-M) and susceptibility testing by Clinical Laboratory Standards Institute (CLSI) disk diffusion method against three cephalosporins (ceftriaxone, ceftazidime, cefepime) and a cephamycin (cefoxitin) among 128 Thai Escherichia coli and 84 Thai Klebsiella pneumoniae clinical isolates. ESBL production was discovered in 62% of E. coli and 43% of K. pneumoniae isolates. All isolates susceptible to ceftriaxone were ESBL-negative. Nearly all isolates non-susceptible to ceftriaxone, ceftazidime and cefepime produced ESBL; the presence of CTX-M genes in the isolates correlated with a ceftriaxone non-susceptible phenotype. Thirty-nine of 83 isolates (47%) of ceftazidime-susceptible E. coli and 50 of 99 isolates (50.5%) of cefepime-susceptible E. coli were ESBL-producing. SHV-type beta-lactamase genes were more prevalent among K. pneumoniae than E. coli isolates. CTX-M was the major ESBL gene harbored by ESBL-producers in both E. coli and K. pneumoniae isolates. Non-CTX-M ESBL-producers were found only among K. pneumoniae isolates. This study reveals an increase in ESBL-producing Enterobacteriaceae among Thai isolates and demonstrates gaps in the current CLSI disk diffusion susceptibility guidelines; it indicates the results of ceftazidime and cefepime disk diffusion susceptibility testing using CLSI criteria should be interpreted with caution.

Errate: Hypoprothrombinemia During Cefmetazole Treatment: A Case Report.

The authors contacted the journal with the following corrections to the published article: 1. Table 1. PT-INR at 6th hospital day. 2.46 is incorrect; Correct: 2.47. 2. Table 2. Line 5, at Purpose of use of CMZ. NA is incorrect; Correct: Infection secondary to Pneumothorax. 3. Table 2. Line 11, at PT-INR, 2.46 is incorrect; Correct: 2.47. 4. References No. 22 and No. 23 are reversed. The authors apologize for these errors and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. Reference: Yuichiro Haba, Hikaru Akizuki, Naoyuki Hashiguchi, Toshio Naito. Hypoprothrombinemia During Cefmetazole Treatment: A Case Report. Am J Case Rep. 2022; 23: e936712. DOI: 10.12659/AJCR.936712.

Assessment of ß-lactams and Carbapenems Antimicrobials Resistance in Klebsiella Oxytoca Isolated from Patients with Urinary Tract Infections in Najaf, Iraq.

Antimicrobial resistance is becoming an arising global issue. Until recent years, more than 50% of commercially available antibiotics were ß-lactam. Pathogenic bacteria which are resistant to antibiotics include all ß-lactams except for cephamycin and carbapenems. This study aimed to evaluate some ß-lactams and carbapenems antimicrobials resistance in Klebsiella oxytoca. In total, 177 urinary tract infection samples were collected for the purposes of the study. Isolates were identified using morphological features and routine biochemical testing. All isolates were tested for susceptibility to 11 antibiotics using the usual disc diffusion method. The result showed that 155 (87.57%) and 20 (11.29%) out of 177 collected urine samples were gram-negative bacterial isolates and gram-positive bacterial isolates, respectively. The findings also showed that there were two samples (1.12 %) with no growth. The results proved no susceptibility to Ampicillin, Cloxacillin, Ceftazidime, Penicillin, Piperacillin with a resistance rate of 100%.

Complete genome sequence of Streptomyces cattleya NRRL 8057, a producer of antibiotics and fluorometabolites.

Streptomyces cattleya, a producer of the antibiotics thienamycin and cephamycin C, is one of the rare bacteria known to synthesize fluorinated metabolites. The genome consists of two linear replicons. The genes involved in fluorine metabolism and in the biosynthesis of the antibiotic thienamycin were mapped on both replicons.

Mutational analysis of the thienamycin biosynthetic gene cluster from Streptomyces cattleya.

The generation of non-thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces cattleya and their involvement in thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of thienamycin production, whereas a 2- to 3-fold increase in thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for thienamycin biosynthesis, as thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydrothienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-thienamycin-producing mutants, lead to a proposal for some enzymatic steps during thienamycin assembly.

Correlation between cephamycin consumption and the incidence of antimicrobial resistance in Acinetobacter baumannii at a university hospital in China from 2001 to 2009.

OBJECTIVES: To investigate the correlation between cephamycin consumption and the prevalence of antimicrobial resistance in Acinetobacter baumannii. METHODS: Cephamycins consumption was expressed as defined daily dose (DDD) per 1,000 patient days by World Health Organization (WHO) Anatomical Therapeutic Chemical (ATC) classification index from 2001 to 2009. The incidences of antimicrobial resistance in A. baumannii were calculated using WHONET 5.4 software in the Microbiology Department. Correlation coefficient was used for statistical analysis. RESULTS: The results showed that cefmetazole and total cephamycin consumption (i.e., cefmetazole, cefoxitin, cefminox) both positively correlated with the percentages of A. baumannii resistance to piperacillin/tazobactam, ceftazidime, cefepime, imipenem/cilastatin, amikacin, levofloxacin, meropenem, respectively, these antimicrobial agents involved beta-lactams, carbapenems, aminoglycosides and fluroquinolones. In addition, the A. baumannii resistance rates of piperacillin/tazobactam, ceftazidime, cefepime, imipenem/cilastatin, meropenem, amikacin, levofloxacin were associated with the A. baumannii resistance rates of a number of antimicrobial drugs. This finding indicated the possible cross-resistances in four different classes of antimicrobial drugs. It could be due to multidrug resistance in A. baumannii. CONCLUSIONS: The cephamycin consumption was significantly related to the prevalence of the antimicrobial drug resistance and might be correlated with multidrug resistance in A. baumannii. Hence, the prescription of cephamycin should be reduced and optimized in order to avoid the rapid increase of antimicrobial resistance in A. baumannii.

A metallo-ß-lactamase enzyme for internal detoxification of the antibiotic thienamycin.

Thienamycin, the first representative of carbapenem antibiotics was discovered in the mid-1970s from soil microorganism, Streptomyces cattleya, during the race to discover inhibitors of bacterial peptidoglycan synthesis. Chemically modified into imipenem (N-formimidoyl thienamycin), now one of the most clinically important antibiotics, thienamycin is encoded by a thienamycin gene cluster composed of 22 genes (thnA to thnV) from S. cattleya NRRL 8057 genome. Interestingly, the role of all thn-genes has been experimentally demonstrated in the thienamycin biosynthesis, except thnS, despite its annotation as putative ß-lactamase. Here, we expressed thnS gene and investigated its activities against various substrates. Our analyses revealed that ThnS belonged to the superfamily of metallo-ß-lactamase fold proteins. Compared to known ß-lactamases such as OXA-48 and NDM-1, ThnS exhibited a lower affinity and less efficiency toward penicillin G and cefotaxime, while imipenem is more actively hydrolysed. Moreover, like most MBL fold enzymes, additional enzymatic activities of ThnS were detected such as hydrolysis of ascorbic acid, single strand DNA, and ribosomal RNA. ThnS appears as a MBL enzyme with multiple activities including a specialised ß-lactamase activity toward imipenem. Thus, like toxin/antitoxin systems, the role of thnS gene within the thienamycin gene cluster appears as an antidote against the produced thienamycin.

Proteomic analysis of a hom-disrupted, cephamycin C overproducing Streptomyces clavuligerus.

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.