Biological Effects of Quinolones: A Family of Broad-Spectrum Antimicrobial Agents.

Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.

Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA.

The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the "replication fork trap" region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the nonpermissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15 to 24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

Stable-Isotope-Informed, Genome-Resolved Metagenomics Uncovers Potential Cross-Kingdom Interactions in Rhizosphere Soil.

The functioning, health, and productivity of soil are intimately tied to a complex network of interactions, particularly in plant root-associated rhizosphere soil. We conducted a stable-isotope-informed, genome-resolved metagenomic study to trace carbon from Avena fatua grown in a 13CO2 atmosphere into soil. We collected paired rhizosphere and nonrhizosphere soil at 6 and 9 weeks of plant growth and extracted DNA that was then separated by density using ultracentrifugation. Thirty-two fractions from each of five samples were grouped by density, sequenced, assembled, and binned to generate 55 unique bacterial genomes that were ≥70% complete. We also identified complete 18S rRNA sequences of several 13C-enriched microeukaryotic bacterivores and fungi. We generated 10 circularized bacteriophage (phage) genomes, some of which were the most labeled entities in the rhizosphere, suggesting that phage may be important agents of turnover of plant-derived C in soil. CRISPR locus targeting connected one of these phage to a Burkholderiales host predicted to be a plant pathogen. Another highly labeled phage is predicted to replicate in a Catenulispora sp., a possible plant growth-promoting bacterium. We searched the genome bins for traits known to be used in interactions involving bacteria, microeukaryotes, and plant roots and found DNA from heavily 13C-labeled bacterial genes thought to be involved in modulating plant signaling hormones, plant pathogenicity, and defense against microeukaryote grazing. Stable-isotope-informed, genome-resolved metagenomics indicated that phage can be important agents of turnover of plant-derived carbon in soil. IMPORTANCE Plants grow in intimate association with soil microbial communities; these microbes can facilitate the availability of essential resources to plants. Thus, plant productivity commonly depends on interactions with rhizosphere bacteria, viruses, and eukaryotes. Our work is significant because we identified the organisms that took up plant-derived organic C in rhizosphere soil and determined that many of the active bacteria are plant pathogens or can impact plant growth via hormone modulation. Further, by showing that bacteriophage accumulate CO2-derived carbon, we demonstrated their vital roles in redistribution of plant-derived C into the soil environment through bacterial cell lysis. The use of stable-isotope probing (SIP) to identify consumption (or lack thereof) of root-derived C by key microbial community members within highly complex microbial communities opens the way for assessing manipulations of bacteria and phage with potentially beneficial and detrimental traits, ultimately providing a path to improved plant health and soil carbon storage.

The regulation of DNA supercoiling across evolution.

DNA supercoiling controls a variety of cellular processes, including transcription, recombination, chromosome replication, and segregation, across all domains of life. As a physical property, DNA supercoiling alters the double helix structure by under- or over-winding it. Intriguingly, the evolution of DNA supercoiling reveals both similarities and differences in its properties and regulation across the three domains of life. Whereas all organisms exhibit local, constrained DNA supercoiling, only bacteria and archaea exhibit unconstrained global supercoiling. DNA supercoiling emerges naturally from certain cellular processes and can also be changed by enzymes called topoisomerases. While structurally and mechanistically distinct, topoisomerases that dissipate excessive supercoils exist in all domains of life. By contrast, topoisomerases that introduce positive or negative supercoils exist only in bacteria and archaea. The abundance of topoisomerases is also transcriptionally and post-transcriptionally regulated in domain-specific ways. Nucleoid-associated proteins, metabolites, and physicochemical factors influence DNA supercoiling by acting on the DNA itself or by impacting the activity of topoisomerases. Overall, the unique strategies that organisms have evolved to regulate DNA supercoiling hold significant therapeutic potential, such as bactericidal agents that target bacteria-specific processes or anticancer drugs that hinder abnormal DNA replication by acting on eukaryotic topoisomerases specialized in this process. The investigation of DNA supercoiling therefore reveals general principles, conserved mechanisms, and kingdom-specific variations relevant to a wide range of biological questions.

The bacteriophage LUZ24 "Igy" peptide inhibits the Pseudomonas DNA gyrase.

The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones. Despite it being a valuable antibiotics target, resistance emergence by pathogens including Pseudomonas aeruginosa are proving problematic. Here, we describe Igy, a peptide inhibitor of gyrase, encoded by Pseudomonas bacteriophage LUZ24 and other members of the Bruynoghevirus genus. Igy (5.6 kDa) inhibits in vitro gyrase activity and interacts with the P. aeruginosa GyrB subunit, possibly by DNA mimicry, as indicated by a de novo model of the peptide and mutagenesis. In vivo, overproduction of Igy blocks DNA replication and leads to cell death also in fluoroquinolone-resistant bacterial isolates. These data highlight the potential of discovering phage-inspired leads for antibiotics development, supported by co-evolution, as Igy may serve as a scaffold for small molecule mimicry to target the DNA gyrase complex, without cross-resistance to existing molecules.

DNA damage checkpoint activation affects peptidoglycan synthesis and late divisome components in Bacillus subtilis.

During normal DNA replication, all cells encounter damage to their genetic material. As a result, organisms have developed response pathways that provide time for the cell to complete DNA repair before cell division occurs. In Bacillus subtilis, it is well established that the SOS-induced cell division inhibitor YneA blocks cell division after genotoxic stress; however, it remains unclear how YneA enforces the checkpoint. Here, we identify mutations that disrupt YneA activity and mutations that are refractory to the YneA-induced checkpoint. We find that YneA C-terminal truncation mutants and point mutants in or near the LysM peptidoglycan binding domain render YneA incapable of checkpoint enforcement. In addition, we develop a genetic method which isolated mutations in the ftsW gene that completely bypassed checkpoint enforcement while also finding that YneA interacts with late divisome components FtsL, Pbp2b, and Pbp1. Characterization of an FtsW variant resulted in considerably shorter cells during the DNA damage response indicative of hyperactive initiation of cell division and bypass of the YneA-enforced DNA damage checkpoint. With our results, we present a model where YneA inhibits septal cell wall synthesis by binding peptidoglycan and interfering with interaction between late arriving divisome components causing DNA damage checkpoint activation.

The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader.

In many bacteria and eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+(ATPases Associated with various cellular Activities) ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of Escherichia coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We have identified a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate that elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as in polymerase clamp loading and certain classes of DNA transposases.

Coordination between nucleotide excision repair and specialized polymerase DnaE2 action enables DNA damage survival in non-replicating bacteria.

Translesion synthesis (TLS) is a highly conserved mutagenic DNA lesion tolerance pathway, which employs specialized, low-fidelity DNA polymerases to synthesize across lesions. Current models suggest that activity of these polymerases is predominantly associated with ongoing replication, functioning either at or behind the replication fork. Here we provide evidence for DNA damage-dependent function of a specialized polymerase, DnaE2, in replication-independent conditions. We develop an assay to follow lesion repair in non-replicating Caulobacter and observe that components of the replication machinery localize on DNA in response to damage. These localizations persist in the absence of DnaE2 or if catalytic activity of this polymerase is mutated. Single-stranded DNA gaps for SSB binding and low-fidelity polymerase-mediated synthesis are generated by nucleotide excision repair (NER), as replisome components fail to localize in the absence of NER. This mechanism of gap-filling facilitates cell cycle restoration when cells are released into replication-permissive conditions. Thus, such cross-talk (between activity of NER and specialized polymerases in subsequent gap-filling) helps preserve genome integrity and enhances survival in a replication-independent manner.

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts.

Lsr2, a nucleoid-associated protein influencing mycobacterial cell cycle.

Nucleoid-associated proteins (NAPs) are responsible for maintaining highly organized and yet dynamic chromosome structure in bacteria. The genus Mycobacterium possesses a unique set of NAPs, including Lsr2, which is a DNA-bridging protein. Importantly, Lsr2 is essential for the M. tuberculosis during infection exhibiting pleiotropic activities including regulation of gene expression (mainly as a repressor). Here, we report that deletion of lsr2 gene profoundly impacts the cell morphology of M. smegmatis, which is a model organism for studying the cell biology of M. tuberculosis and other mycobacterial pathogens. Cells lacking Lsr2 are shorter, wider, and more rigid than the wild-type cells. Using time-lapse fluorescent microscopy, we showed that fluorescently tagged Lsr2 forms large and dynamic nucleoprotein complexes, and that the N-terminal oligomerization domain of Lsr2 is indispensable for the formation of nucleoprotein complexes in vivo. Moreover, lsr2 deletion exerts a significant effect on the replication time and replisome dynamics. Thus, we propose that the Lsr2 nucleoprotein complexes may contribute to maintaining the proper organization of the newly synthesized DNA and therefore influencing mycobacterial cell cycle.

Promutagenic bypass of 7,8-dihydro-8-oxoadenine by translesion synthesis DNA polymerase Dpo4.

Reactive oxygen species induced by ionizing radiation and metabolic pathways generate 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major forms of oxidative damage. The mutagenicity of oxoG, which promotes G to T transversions, is attributed to the lesion's conformational flexibility that enables Hoogsteen base pairing with dATP in the confines of DNA polymerases. The mutagenesis mechanism of oxoA, which preferentially causes A to C transversions, remains poorly characterized. While structures for oxoA bypass by human DNA polymerases are available, that of prokaryotic DNA polymerases have not been reported. Herein, we report kinetic and structural characterizations of Sulfolobus solfataricus Dpo4 incorporating a nucleotide opposite oxoA. Our kinetic studies show oxoA at the templating position reduces the replication fidelity by ∼560-fold. The catalytic efficiency of the oxoA:dGTP insertion is ∼300-fold greater than that of the dA:dGTP insertion, highlighting the promutagenic nature of oxoA. The relative efficiency of the oxoA:dGTP misincorporation is ∼5-fold greater than that of the oxoG:dATP misincorporation, suggesting the mutagenicity of oxoA is comparable to that of oxoG. In the Dpo4 replicating base pair site, oxoA in the anti-conformation forms a Watson-Crick base pair with an incoming dTTP, while oxoA in the syn-conformation assumes Hoogsteen base pairing with an incoming dGTP, displaying the dual coding potential of the lesion. Within the Dpo4 active site, the oxoA:dGTP base pair adopts a Watson-Crick-like geometry, indicating Dpo4 influences the oxoA:dGTP base pair conformation. Overall, the results reported here provide insights into the miscoding properties of the major oxidative adenine lesion during translesion synthesis.

Two components of DNA replication-dependent LexA cleavage.

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA-RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.

How similar or dissimilar cells are produced by bacterial cell division?

DNA replication, segregation and cell division are vital processes and require an interplay of multiple proteins. These processes are highly conserved across bacteria yet similar or dissimilar progeny are produced after cell division. This review describes the bacterial cell division in considerable detail. This includes studies on model microorganisms which produce similar progeny such as Escherichia coli and Vibrio cholerae, and dissimilar progeny such as sporulating Bacillus subtilis, Actinobacteria, Caulobacter crescentus etc. The mechanism of symmetric and asymmetric cell division and its regulation has also been discussed.

A Comprehensive View of Translesion Synthesis in Escherichia coli.

The lesion bypass pathway, translesion synthesis (TLS), exists in essentially all organisms and is considered a pathway for postreplicative gap repair and, at the same time, for lesion tolerance. As with the saying "a trip is not over until you get back home," studying TLS only at the site of the lesion is not enough to understand the whole process of TLS. Recently, a genetic study uncovered that polymerase V (Pol V), a poorly expressed Escherichia coli TLS polymerase, is not only involved in the TLS step per se but also participates in the gap-filling reaction over several hundred nucleotides. The same study revealed that in contrast, Pol IV, another highly expressed TLS polymerase, essentially stays away from the gap-filling reaction. These observations imply fundamentally different ways these polymerases are recruited to DNA in cells. While access of Pol IV appears to be governed by mass action, efficient recruitment of Pol V involves a chaperone-like action of the RecA filament. We present a model of Pol V activation: the 3' tip of the RecA filament initially stabilizes Pol V to allow stable complex formation with a sliding ß-clamp, followed by the capture of the terminal RecA monomer by Pol V, thus forming a functional Pol V complex. This activation process likely determines higher accessibility of Pol V than of Pol IV to normal DNA. Finally, we discuss the biological significance of TLS polymerases during gap-filling reactions: error-prone gap-filling synthesis may contribute as a driving force for genetic diversity, adaptive mutation, and evolution.

The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates.

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

Incorporation characteristics of exogenous 15N-labeled thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine into bacterial DNA.

Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.

Formation of microbial products by activated sludge in the presence of a metabolic uncoupler o-chlorophenol in long-term operated sequencing batch reactors.

Metabolic uncouplers are widely used for reducing excess sludge in biological wastewater treatment systems. However, the formation of microbial products, such as extracellular polymeric substances, polyhydroxyalkanoate and soluble microbial products by activated sludge in the presence of metabolic uncouplers remains unrevealed. In this study, the impacts of a metabolic uncoupler o-chlorophenol (oCP) on the reduction of activated sludge yield and formation of microbial products in laboratory-scale sequencing batch reactors (SBRs) were evaluated for a long-term operation. The results show the average reduction of sludge yield in the four reactors was 17.40%, 25.80%, 33.02% and 39.50%, respectively, when dosing 5, 10, 15, and 20 mg/L oCP. The oCP addition slightly reduced the pollutant removal efficiency and decreased the formation of soluble microbial products in the SBRs, but stimulated the productions of extracellular polymeric substances and polyhydroxyalkanoate in activated sludge. Furthermore, the significant reduction of electronic transport system activity occurred after the oCP addition. Microbial community analysis of the activated sludge indicates dosing oCP resulted in a decrease of sludge richness and diversity in the SBRs. Hopefully, this study would provide useful information for reducing sludge yield in biological wastewater treatment systems and behaviors of activated sludge in the presence of uncouplers.

E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication.

During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3'-terminus of the primer provides four C-termini for protein-protein interactions.

The antibiotic activity and mechanisms of active metabolites (Streptomyces alboflavus TD-1) against Ralstonia solanacearum.

OBJECTIVES: In order to elucidate the antibacterial activity and mechanism of S. alboflavus TD-1 active metabolites, the minimal inhibitory concentration of R. solanacearum and other effects on cell wall, cell membrane, nucleic acid, protein and cell morphology were studied. Besides, based on LCMS-IT-TOF, the active metabolites of S. alboflavus TD-1 were preliminarily analyzed. RESULTS: In this study, We found that the active metabolites had obvious inhibitory effect on R. solanacearum, and the minimal inhibitory concentration (MIC) of R. solanacearum was 3.125 mg/mL. And the treatment of 10 mg/mL active metabolites can increase the permeability of R. solanacearum membranes, destroy the cell wall integrity, inhibit the synthesis of bacterial nucleic acids and proteins, and cause leakage of bacterial nucleic acids and proteins, obstruct the normal expression of proteins and destroy their bacterial morphology. At the same time, We speculated the molecular weights corresponding to the six compounds were 618, 615, 615, 615, 646, 646, respectively among the active metabolites, and it was found that were highly unstable. CONCLUSIONS: The active metabolites produced by S. alboflavus TD-1 liquid fermentation contain components that can significant inhibitory effects on R. solanacearum. It had the potential to develop biocontrol agents against bacterial wilt and be a kind potential sources for the preparation of functional anti-pathogenic microbial agents.

Plasmid production and purification: An integrated experiment-based biochemistry and biotechnology laboratory course.

This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one-week period includes the protocols for plasmid pVAX1-LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1-LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1-LacZ plasmid. Lastly, they perform the purification of pVAX1-LacZ supercoiled isoform by L-histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638-643, 2019.