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Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks., Competing Interests: The authors have declared that no competing interests exist.

After the first description of OXA-48 type carbapenemase, it has become endemic in Europe, Mediterranean and North African countries in a short time. OXA-48 carbapenemase is the most difficult type to determine and accurate diagnosis is crucial especially in endemic areas.The CarbaNP test was described as a rapid phenotypic evaluation method of carbapenemases activity. Sensitivity and specifity of this test were high within all carbapenemases genes. In our study, we evaluated the efficacy of CarbaNP test in routine laboratories located in an endemic area of OXA-48 producing Enterobacterales.A total of 53 Enterobacterales isolates were included in this study. Antimicrobial susceptibility of the isolates to imipenem, meropenem and ertapenem was determined. Polymerase Chain Reaction (PCR) was carried out for the detection of carbapenemases genes (blaKPC, blaNDM, blaBIC, blaIMP, blaVIM, blaSPM, blaAIM, blaDIM, blaGIM, blaSIM, and blaOXA-48). The Carba NP test was performed as in the protocol described previously.Altogether 31 isolates (58.4%) were blaOXA-48 positive (18 Klebsiella pneumoniae, 8 Escherichia coli, 2 Serratia marcescens, 1 Enterobacter aerogenes, 1 Pantoea agglomerans and 1 Morganella morganii). Among these isolates 3 (5.6%) and 2 (3.7%) isolates were also positive for blaVIM and blaSPM, respectively.The sensitivity and specifity of CarbaNP test were found 64.5, and 68.2% respectively. It was observed that determination of positive isolates is hard to distinguish and subjective.The CarbaNP test has suboptimal results and low of sensitivity and specifity for detection of OXA-48 producing Enterobacterales, and not suitable for detection of blaOXA-48 positive isolates in routine laboratories in endemic areas.

Resistance to carbapenems due to metallo-beta-lactamase NDM-1 was first described in Brazil in 2013. To date, only a few scattered reports of the prevalence of NDM-1 in the country have been reported, and most of them indicated a very low prevalence of this metalloenzyme. In the present study, we report a steady increase in the frequency of NDM among Enterobacterales resistant to carbapenems in a tertiary care hospital in southern Brazil. Carbapenemase genes were evaluated by multiplex real-time polymerase chain using high-resolution melting analysis among 3501 isolates of 8 different species of Enterobacterales recovered from January 2015 to May 2020. The bla KPC-like was identified in 3003 isolates (85.8%) and the bla NDM-like was the second most common gene (351 isolates-10%). There was a steady increase in frequency of bla NDM-like , from 4.2% in 2015 to 24% in 2020. The increase of bla NDM frequency raises an important matter as novel therapeutic options are currently very limited for the treatment of patients infected by bacteria carrying the bla NDM .

Autotrophic or mixotrophic use of one-carbon (C1) compounds is gaining importance for sustainable bioproduction. In an effort to integrate the reductive glycine pathway (rGP) as a highly promising pathway for the assimilation of CO 2 and formate, genes coding for glycine synthase system from Gottschalkia acidurici were successfully introduced into Clostridium pasteurianum, a non-model host microorganism with industrial interests. The mutant harboring glycine synthase exhibited assimilation of exogenous formate and reduced CO 2 formation. Further metabolic data clearly showed large impacts of expression of glycine synthase on the product metabolism of C. pasteurianum. In particular, 2-oxobutyrate (2-OB) was observed for the first time as a metabolic intermediate of C. pasteurianum and its secretion was solely triggered by the expression of glycine synthase. The perturbation of C1 metabolism is discussed regarding its interactions with pathways of the central metabolism, acidogenesis, solventogenesis, and amino acid metabolism. The secretion of 2-OB is considered as a consequence of metabolic and redox instabilities due to the activity of glycine synthase and may represent a common metabolic response of Clostridia in enhanced use of C1 compounds., (© 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

An endo-1,4-β-mannanase gene (manB) from a Bacillus pumilus Nsic-2 grown in a stinky tofu emulsion was cloned and expressed in Pichia pastoris GS115. After characterized, the endo-1,4-β-mannanase (manB) show maximum activity at pH 6.0 and 50 °C with LBG as substrate and perform high stability at a range of pH 6-8. After applying for a shake flask fermentation, the specific activity of manB reached 3462 U/mg. To produce mannose, the soybean meal (SBM) was pretreated by biological fermentation for 11 days with Penicillium brevicompactum, and then hydrolyzed by manB. As a result, mannose yield reached 3.58 g per 1 kg SBM which indicated that 0.358% SBM was converted into mannose after hydrolyzation, and mean a total 20% mannan of SBM converting into mannose, while the control group demonstrated only 1.78% conversion. An effective β-mannanase for the bioconversion of mannan-rich biomasses and an efficient method to produce mannose with soybean meal were introduced.

Biodiesel biosynthesis with enzymatic transesterification is considered green, sustainable, and environmentally friendly method. Lipase from Burkholderia cepacia G63 has excellent catalytic properties in biodiesel production. Lipase chaperones promote secretion and folding of enzymes, thereby enhancing enzymatic activity. In the current study, heterologous co-expression of lipase (lipA) and chaperone (lipB) was achieved in Escherichia coli through codon optimization. The enzymatic activity of purified and renatured lipAB was 2080.23 ± 19.18 U/g at 50 °C and pH 8.0. Moreover, lipAB showed increased resistance to pH and temperature changes, and lipAB retained stable catalytic properties after treatment with metal ions, organic solvents, and surfactants, namely Mg 2+ , methanol, and Triton-100X. Besides, using recombinant lipase lipAB as catalysts, biodiesel was synthesized using rapeseed oil under 50 °C for 72 h with a yield of 90.23%. Thus, the current study confirmed that co-expression of lipase and its chaperone is an effective strategy to enhance enzyme activity and improve the biochemical profile, meanwhile, showing that lipAB is a promising biocatalyst for biodiesel production.

The NitroSpeed-Carba NP test was used to rapidly detect and discriminate between the different types of carbapenemases (classes A, B, and D) within 30 minutes among a collection of 202 Pseudomonas sp. strains (mostly Pseudomonas aeruginosa). A total of 99 carbapenemase-(including enzymes exhibiting weak carbapenemase activity such as several Guyana Extended-Spectrum (GES)-ß-lactamases) and 103 non-carbapenemase producers were tested, and the overall specificity and sensitivity were 100% and 99%, respectively. The NitroSpeed-Carba NP test is a rapid, specific, sensitive, and easy-to-implement technique for identification of carbapenemase-producing Pseudomonas spp., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5' untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5'-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3'-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 10 8 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca 2+ /mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca 2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Background: Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium., Results: We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium., Conclusions: Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its Δ lasR -derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain. IMPORTANCE Pseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections., (Copyright © 2021 American Society for Microbiology.)

A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485 nm. Site-directed saturation mutagenesis libraries of D-threonine aldolase from Alcaligenes xylosoxidans CGMCC 1.4257 (AxDTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants AxDTA D321C and AxDTA N101G were identified. Substrate specificity of AxDTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore, AxDTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.

Glucan branching enzymes (GBEs, EC 2.4.1.18) catalyze the formation of α-1,6-linked branch in starch, which is important for the starch modification with prospective properties. In this study, the aqGBE gene encoding an efficient glucan branching enzyme was cloned from Aquabacterium sp. strain A7-Y and successfully expressed in Escherichia coli BL21 (DE3). The specific activity of the purified recombinant enzyme rAqGBE was 2850 U/mg with potato starch as the optimal substrate, and the K m and V max values of rAqGBE were 1.18 mg/mL and 588.2 μmol/min/mg, respectively. Enzymological characterization showed that rAqGBE exhibits its optimal activity under the condition of 40 °C and pH 7.0, respectively, which is independent of calcium ions. Otherwise, rAqGBE-treated potato starch showed different chain length distribution compared with control, the numbers of short chains (degree of polymerization, DP < 7) and long chains (DP > 25) increased from 4.5% to 9.6% and 6.1%-15.7% after enzymatic treatment, respectively. In starch anti-ageing assay, with minimum usage of 0.8 mg rAqGBE per g starch, the rAqGBE-treated potato starch exhibited reduced retrogradation properties. Our results indicate that the branching enzyme AqGBE may therefore be a promising tool for the enzymatic modification of starch., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Extracellular proteolytic extracts from the haloalkalitolerant strain Alkalihalobacillus patagoniensis PAT 05 T have proved highly efficient to reduce wool felting, as part of an ecofriendly treatment suitable for organic wool. In the present study, we identified the extracellular proteases produced by PAT 05 T and we optimized its growth conditions for protease production through statistical methods. A total of 191 proteins were identified in PAT 05 T culture supernatants through mass spectrometry analysis. Three of the 6 detected extracellular proteases belonged to the serine-endopeptidase family S8 (EC 3.4.21); two of them showed 86.3 and 67.9% identity with an alkaline protease from Bacillus alcalophilus and another one showed 50.4% identity with Bacillopeptidase F. The other 3 proteases exhibited 55.3, 49.4 and 61.1% identity with D-alanyl-D-alanine carboxypeptidase DacF, D-alanyl-D-alanine carboxypeptidase DacC and endopeptidase LytE, respectively. Using a Fractional Factorial Design followed by a Central Composite Design optimization, a twofold increase in protease production was reached. NaCl concentration was the most influential factor on protease production. The usefulness of PAT 05 T extracellular proteolytic extracts to reduce wool felting was possible associated with the activity of the serine-endopeptidases closely related to highly alkaline keratinolytic proteases. The other identified proteases could cooperate, improving protein hydrolysis. This study provided valuable information for the exploitation of PAT 05 T proteases which have potential for the valorization of organic wool as well as for other industrial applications.

Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with chronic infection and antibiotic treatment failure. Toxin-antitoxin systems have been linked to persister cell formation but the molecular mechanisms leading to bacterial persistence are mostly unknown. Here, we show that SprF1, a type I antitoxin, associates with translating ribosomes from the major human pathogen Staphylococcus aureus to reduce the pathogen's overall protein synthesis during growth. Under hyperosmotic stress, SprF1 levels increase due to enhanced stability, accumulate on polysomes and attenuate protein synthesis. Using an internal 6-nucleotide sequence on its 5'-end, SprF1 binds ribosomes and interferes with initiator transfer RNA binding, thus reducing translation initiation. An excess of messenger RNA displaces the ribosome-bound antitoxin, freeing the ribosomes for new translation cycles; however, this RNA antitoxin can also displace ribosome-bound mRNA. This translation attenuation mechanism, mediated by an RNA antitoxin, promotes antibiotic persister cell formation. The untranslated SprF1 is a dual-function RNA antitoxin that represses toxin expression by its 3'-end and fine-tunes overall bacterial translation via its 5'-end. These findings demonstrate a general function for a bacterial RNA antitoxin beyond protection from toxicity. They also highlight an RNA-guided molecular process that influences antibiotic persister cell formation.

Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD + , suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gene expression is tightly controlled by transcription factors and RNA regulatory elements, including trans-acting small RNAs, cis-regulatory riboswitches and ribosome-dependent ribo-regulators. In the present study, we demonstrated that a ribosome-dependent ribo-regulator and two mistranslation products co-regulate rppA (encoding a ribosomal protection protein) expression in Bacillus thuringiensis BMB171. The leader RNA of the rppA gene controls rppA expression via translation of leader ORF1 resident in its sequence. In the presence of chloramphenicol, a +1 frameshift product (ORF2) and a stop codon readthrough product (ORF3) of ORF1 emerged. ORF3 exerted a negative effect on rppA expression. By contrast, the ORF2 promoted rppA expression. The regulation mode identified in the present study will lead to a deeper understanding of bacterial gene expression., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P) + ) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cell growth and product formation are two critical processes in polysaccharide welan biosynthesis, but the conflict between them is often encountered. In this study, a temperature-dependent strategy was designed for two-stage welan production through overexpressing heat shock proteins in Sphingomonas sp. The first stage was cell growth phase with higher TCA cycle activity at 42 °C; the second stage was welan formation phase with higher precursor synthesis pathway activity at 37 °C. The highest welan concentration 37.5 g/L was achieved after two-stage process. Ultimately, this strategy accumulated welan yield of 79.2 g/100 g glucose and productivity of 0.62 g/L/h at 60 h, which were the best reported results so far. The duration of fermentation was shortened. Besides, rheological behavior of welan gum solutions remained stable at wide range of temperature, pH, and NaCl. These results indicated that this approach efficiently improved welan synthesis.

The principal etiological agent of human dental caries, Streptococcus mutans is a multi-virulent pathogen that can transform commensal oral microbial community to plaque biofilms. Major virulence factors that are associated with the cariogenicity of S. mutans include adhesion, acidogenicity and acidurity. All these pathogenic traits coordinate and alter the dental plaque ecology which provide room for interaction with other similar acidogenic and aciduric bacteria. This cariogenic flora increases the possibility of enamel demineralization which headway to caries development. The present study was aimed at evaluating the antimicrobial and antiinfective potential of a lichen secondary metabolite usnic acid (UA) against S. mutans. Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC) and growth kinetics were evaluated to determine the antimicrobial potential of UA against S. mutans. UA at 5 µg mL -1 and 10 µg mL -1 concentration were considered as MIC and MBC respectively. Effect on biofilm formation was microscopically assessed and found to be reduced in a concentration dependent manner. Gene expression of gtfB, gtfC, gtfD, vicR, ComDE and smu0630 was found to be downregulated upon treatment with sub-MIC of UA. Acidogenicity, acidurity, eDNA synthesis and response to oxidative stress were found to be attenuated by the influence of UA. It was also demonstrated to act on preformed mature biofilm of S. mutans. Moreover, UA was shown to possess very low frequency to acquire spontaneous resistance development in S. mutans. Besides, no morphological aberrations or toxic effect was instigated by UA in the human buccal epithelial cells as well as to the oral commensals. Altogether, these results demonstrate the therapeutic potential of usnic acid in the treatment of S. mutans infection.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

Optimization of cellulase production by Bacillus subtilis subsp. subtilis JJBS300 resulted in maximum cellulase (CMCase 9.7 U/g substrate) using wheat bran and rice straw in 1:1 ratio at substrate to moisture ratio of 1:3 at 35 °C and pH 4.0 after 48 h. Partially purified cellulase of B. subtilis subsp. subtilis showed optimal activity at 50 °C and pH 5.0. Among the metal ions, Na + , Ca 2+ and Fe 2+ stimulated the cellulase activity. Glutaraldehyde and 1-butanol also enhanced the cellulase activity as compared to other solvents. Bacterial cellulase hydrolyzed ammonia-pretreated rice straw more efficiently as compared to sodium-carbonate pretreated and untreated biomass. Optimization of saccharification of untreated and pretreated (sodium carbonate and ammonia) rice straw by bacterial cellulase resulted in high liberation of reducing sugars with enzyme dose of 100 U/g substrate (221 mg/g substrate) at pH 5.0 (103 mg/g substrate) and 50 °C (142 mg/g substrate) after 6 h in ammonia-pretreated rice straw. Furthermore, liberation of reducing sugars increased with incubation time showing maximum reducing sugars (171 mg/g substrate) after 24 h in ammonia-pretreated rice straw. HPLC analysis of enzymatic hydrolysate of ammonia-pretreated rice straw verified the ability of bacterial cellulase in liberation of various monomeric and oligomeric sugars.

Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m 3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m 3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD 600 ) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD 600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD 600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.

Organic solvent-tolerant proteases have many applications in the synthesis of peptides. In this study, we have developed a low-cost and convenient method to produce highly concentrated organic solvent-tolerant protease. Organic solvent tolerant protease (OSP) gene from Bacillus sphaericus DS11 was cloned and expressed in Bacillus subtilis WB800. The optimum pH of the recombinant protease was 9.0. The optimum temperature of the recombinant protease was 40 °C. The recombinant protease was purified by ethanol with the yield of (87.33%). The yield of OSP enriched by ethanol was higher than that of by Ni-chelating affinity chromatography, which indicated that precipitation of the recombinant OSP with ethanol is a relatively low-cost and fast method for organic solvent -tolerant protease preparation. These results showed that this enzyme could be very useful in different industrial applications.

Bacillus licheniformis 2709 is the major alkaline protease producer, which has great potential value of industrial application, but how the high-producer can be regulated rationally is still not completely understood. It's meaningful to understand the metabolic processes during alkaline protease production in industrial fermentation medium. Here, we collected the transcription database at various enzyme-producing stages (preliminary stage, stable phase and decline phase) to specifically research the synthesized and regulatory mechanism of alkaline protease in B. licheniformis. The RNA-sequencing analysis showed differential expression of numerous genes related to several processes, among which genes correlated with regulators were concerned, especially the major differential gene abrB on enzyme (AprE) synthesis was investigated. It was further verified that AbrB is a repressor of AprE by plasmid-mediated over-expression due to the severely descending enzyme activity (11,300 U/mL to 2695 U/mL), but interestingly it is indispensable for alkaline protease production because the enzyme activity of the null abrB mutant was just about 2279 U/mL. Thus, we investigated the aprE transcription by eliminating the theoretical binding site (TGGAA) of AbrB protein predicated by computational strategy, which significantly improved the enzyme activity by 1.21-fold and gene transcription level by 1.77-fold in the mid-log phase at a cultivation time of 18 h. Taken together, it is of great significance to improve the production strategy, control the metabolic process and oriented engineering by rational molecular modification of regulatory network based on the high throughput sequencing and computational prediction., Competing Interests: Declaration of competing interest The authors declare that there is not any competing interest., (Copyright © 2020. Published by Elsevier B.V.)

Extracellular protease production by a novel strain, Bacillus sp. EBTA6, has been optimized by using central composite design of response surface methodology and properties and industrial applications of crude enzyme have been investigated. Three independent variables (temperature, pH and yeast extract concentration) chosen in the experimental design were significant terms and reduced cubic model fit with the design at p  < 0.0001 level. The recommended temperature, pH and yeast extract concentration were 30 °C, 8, and 15 g/L, respectively. Crude enzyme displayed activity over a wide pH and temperature ranges having the optimum at 50-60 °C and pH 8. It was quite stable at high pH values and at 50 °C. Amongst the metal ions (Mg + , Cu 2+ , Ca 2+ , Zn 2+ , K 2+ , and Sn 2+ ), Ca 2+ enhanced the activity and the others either decreased or did not change it. The enzyme activity was reduced by phenyl-methyl-sulfonyl fluoride (PMSF), and ethylene diamine tetra acetic acid (EDTA). The results revealed that the protease was serine alkaline type. Tween 20 and Tween 80 did not inhibit the enzyme, however, sodium dodecyl sulfate (SDS), reduced it by 39%. It completely removed blood stain in 20 min and coagulated milk in the presence of CaCl 2 .

Background: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent., Objective: This study aimed to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide., Methods: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A) were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain., Results: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and it exhibited larvicidal activity against Aedes aegypti., Conclusion: B. subtilis was successfully developed as a host for the production of secreted Mtx2, and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Heat shock proteins (HSPs) represent a group of important proteins which are produced by all kinds of organisms especially under stressful conditions. DnaK, an Hsp70 homolog in prokaryotes, has indispensable roles when microbes was confronted with stress conditions. However, few data on DnaK from Rhodococcus sp. were available in the literature. In a previous study, we reported that toluene and phenol stress gave rise to a 29.87-fold and 3.93-fold increase for the expression of DnaK from R. ruber SD3, respectively. Thus, we deduced DnaK was in correlation with the organic solvent tolerance of R. ruber SD3., Objective: To elucidate the role of DnaK in the organic solvent tolerance of R. ruber SD3, expression, purification and functional analysis of Dnak from R. ruber SD3 were performed in the present paper., Methods: In this article, DnaK from R. ruber SD3 was heterologously expressed in E. coli BL21(DE3) and purified by affinity chromatography. Functional analysis of DnaK was performed using determination of kinetics, docking, assay of chaperone activity and microbial growth., Results: The recombinant DnaK was rapidly purified by affinity chromatography with the purification fold of 1.9 and the recovery rate of 57.9%. Km, Vmax and Kcat for Dnak from R. ruber SD3 were 80.8 μM, 58.1 nmol/min and 374.3 S -1 , respectively. The recombinant protein formed trimer in vitro, with the calculated molecular weight of 214 kDa. According to in-silico analysis, DnaK interacted with other molecular chaperones and some important proteins in the metabolism. The specific activity of catalase in the presence of recombinant DnaK was 1.85 times or 2.00 times that in the presence of BSA or Tris-HCl buffer after exposure to 54 °C for 1h. E. coli transformant with pET28-dnak showed higher growth than E. coli transformant with pET28 at 43°C and in the presence of phenol, respectively., Conclusion: The biochemical properties and the interaction analysis of DnaK from R. ruber SD3 deepened our understanding of DnaK function. DnaK played an important role in microbial growth when R. ruber was subjected to various stress such as heating and organic solvent., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We investigated the presence of carbapenemases in carbapenem-resistant Pseudomonas aeruginosa isolates, which were collected over a 14-month period in a Turkish hospital, with in-depth molecular characterization of carbapenemase-producing isolates. Among 45 study isolates, 2 isolates were identified as carbapenemase producers by both Carba NP and Carbapenem Inactivation Method tests, and only 1 of them gave a positive result in polymerase chain reaction tests for a carbapenemase gene (bla VIM ). Whole genome sequencing of the 2 isolates revealed the presence of bla VIM-5 gene in an ST308 isolate, while the other one expressed IMP-7 in an ST357 isolate; both STs are considered high-risk clones. The 2 carbapenemase-producing isolates were multidrug resistant, as they harbored other resistance determinants, including a variant of the recently described plasmid-encoded fluoroquinolone resistance determinant crpP gene, crpP-2. We report for the first time P. aeruginosa high-risk clones carrying VIM-5- and IMP-7-type carbapenemases with multiple resistance determinants in Turkey., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

Background: Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative bacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization of the Ot strain into host cells and are suitable resources for vaccine development and tools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter (AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be promising candidates for vaccine development. These proteins are typically large and contain inhibitory domains; therefore, recombinant proteins without such domains have been evaluated for this purpose. However, the expression for recombinant Sca proteins containing the AT domain, which might largely affect their protective role against scrub typhus, has not been analyzed and optimized., Objective: In this study, we optimized expression and purification conditions for individual Ot Sca protein fragments [ScaA (27-1461), ScaC (257-526), ScaD (26-998), and ScaE (35-760)] harboring the AT domain., Methods: To this end, we subcloned sequences of codon-optimized DNA encoding Sca protein fragments into the Escherichia coli expression vector. In addition, the expression condition for individual Sca fragments was optimized, and the proteins were purified using one-step Ni-NTA column method. The purified fractions were re-folded by serial dilution method, followed by BCA quantification and densitometric analysis to estimate the yield and purity of proteins., Results: We prepared platforms for expression of recombinant Sca protein fragments [ScaA (27-1461), ScaC (257-526), ScaD (26-998), and ScaE (35-760)] containing an AT domain without the signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with >70% of purity was ScaC (257-576), ScaE (35-760), ScaD (26-998), and ScaA (27-1461) in order., Conclusion: Our results could be used to develop Sca AT-domain based vaccines and tools for scrub typhus diagnosis with rapid and cost-effective ways., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The vital state variables in marine alkaline protease (MP) fermentation are difficult to measure in real-time online, hardly is the optimal control either. In this article, a dynamic soft sensor modeling method which combined just-in-time learning (JITL) technique and ensemble learning is proposed. First, the local weighted partial least squares algorithm (LWPLS) with JITL strategy is used as the basic modeling method. For further improving the prediction accuracy, the moving window (MW) is used to divide sub-dataset. Then the MW-LWPLS sub-model is built by selecting the diverse sub-datasets according to the cumulative similarity. Finally, stacking ensemble-learning method is utilized to fuse each MW-LWPLS sub-models. The proposed method is applied to predict the vital state variables in the MP fermentation process. The experiments and simulations results show that the prediction accuracy is better compared to other methods.

Vitamin B 12 , cobalamin, is a cobalt-containing ring-contracted modified tetrapyrrole that represents one of the most complex small molecules made by nature. In prokaryotes it is utilised as a cofactor, coenzyme, light sensor and gene regulator yet has a restricted role in assisting only two enzymes within specific eukaryotes including mammals. This deployment disparity is reflected in another unique attribute of vitamin B 12 in that its biosynthesis is limited to only certain prokaryotes, with synthesisers pivotal in establishing mutualistic microbial communities. The core component of cobalamin is the corrin macrocycle that acts as the main ligand for the cobalt. Within this review we investigate why cobalt is paired specifically with the corrin ring, how cobalt is inserted during the biosynthetic process, how cobalt is made available within the cell and explore the cellular control of cobalt and cobalamin levels. The partitioning of cobalt for cobalamin biosynthesis exemplifies how cells assist metalation., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Multi-drug resistance among AmpC β-lactamases producing Escherichia coli isolates is alarming. The study aimed to know the prevalence and presumptive antibiogram of AmpC producing Escherichia coli isolates and to determine the associated risk factors., Methodology: Escherichia coli isolated from various clinical specimens from hospitalized patients during the study period (January 2018- December 2018) were taken for the study. Standard biochemical reactions were used for organism identification. Antibiotic susceptibility testing was done using Kirby-Bauer method as per CLSI guidelines. Cefoxitin resistance was taken as screening tool to detect AmpC producing strains. The phenotypic confirmation was done using modified three-dimensional test. Multiplex PCR was used to detect pAmpC., Results: A total non-duplicate consecutive 470 Escherichia coli were isolated from various clinical specimens of hospitalized patients during the study period. Cefoxitin resistance was observed in 51.9% (244/470). Modified three dimensional test was positive in 115/244 (47.1%) strains. Genotypic characterization of phenotypic positive AmpC strains showed presence of CIT and DHA genes among 33/115 and 19/115 isolates respectively. The overall prevalence of pAmpC producing E. coli was found to be 52/470 (11.1%). Multidrug resistance (MDR) was observed in 42/52 (80.7%) pAmpC strains. Antimicrobial use, prolonged hospitalization and interventions were associated risk factors for AmpC producing isolates., Conclusions: A high prevalence of multidrug resistance among AmpC producing strains suggests plasmid mediated spread of drug resistance in E. coli. Every hospital should formulate and implement infection control policies at-least for the risk group patients to control the dissemination of such microbes as infection prevention is better than infection control., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2020 Rosy Bala, Varsha A Singh, Nitin Gupta, Pue Rakshit.)

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: The dramatic increase in the prevalence and clinical impact of infections caused by Carbapenemase-Producing Bacteria in the nosocomial setting in Latin America represents an emerging challenge to public health. The present study detected carbapenemase-producing Gram-negative bacteria in patients from a Hospital from Venezuela, by phenotypic and genotypic methods., Methodology: The bacterial identification was carried out using conventional methods. The resistance to carbapenems was performed by Kirby-Baüer disk diffusion method, according to CLSI recommendations. The modified Hodge Test, double-disk with phenylboronic acid, double-disk with EDTA and Blue Carba Test were performed to detect phenotypic carbapenemase producers. The carbapenemase-encoding genes blaKPC, blaVIM, blaIMP, blaOXA-2, blaOXA-3, blaOXA-15 and blaOXA-21 were determined., Results: The bacterial species identified were Klebsiella pneumoniae complex (181), Pseudomonas aeruginosa (51), and Acinetobacter baumannii-calcoaceticus complex (119). KPC-type was detected in 40.17% of isolates and VIM-type in 14.53%. KPC-type gene was only identified in K. pneumoniae isolates (77.9%). VIM-type gene was identified in P. aeruginosa (86.27%) and K. pneumoniae isolates (3.87%). There was not detection of IMP-type and OXA-type genes., Conclusions: We found a predominance of K. pneumoniae KPC producers and a high rate of VIM-producing P. aeruginosa. The epidemiology of CPB in Venezuela is rapidly evolving, and enhanced surveillance and reporting are needed across the healthcare continuum., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Liliana Gomez-Gamboa, Armindo Perozo-Mena, Jose Bermudez-Gonzalez, Christty Villavicencio, Jessica Villasmil, Messaria M Ginestre, Jesvy Velasquez.)

A novel cold-active true lipase from Pseudomonas sp. KE38 was cloned, sequencing and expressed in E. coli by degenerate PCR and genome walking technique. The open reading frame of the cloned gene encoded a polypeptide chain of 617 amino acids with a confirmed molecular weight of 64 kD. Phylogenetic analysis of the deduced amino acid sequence of the lipase indicated that it had high similarity with lipases of subfamily Ι.3 of bacterial lipases. Recombinant lipase was purified in denatured form as inclusion bodies, which were then renatured by urea followed by dialysis. Lipase activity was determined titrimetrically using olive oil as substrate. The enzyme showed optimal activity at 25 °C, pH 8.5 and was highly stable in the presence of various metal ions and organic solvents. Low optimal temperature and high activity in the presence of methanol and ethanol make this lipase a potential candidate for transesterification reactions and biodiesel production.

A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo - was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo - achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cyanobacteria are essential for the vast number of compounds they produce and the possible applications in the pharmaceutical, cosmetical, and food industries. As Lyngbya species' characterization is limited in the literature, we characterize this cyanobacterium's growth and biomass. L . purpureum was grown and analyzed under different salinities, culture media, and incubation times to determine the best conditions that favor its cell growth and the general production of proteins, carbohydrates, lipids, and some pigments as phycocyanin and chlorophyll a . In this study, each analyzed biomolecule's highest content was proteins 431.69 mg g -1 , carbohydrates 301.45 mg g -1 , lipids 131.5 mg g -1 , chlorophyll a 4.09 mg g -1 , and phycocyanin 40.4 mg g -1 . These results can provide a general context of the possible uses that can be given to biomass and give an opening to investigate possible biocompounds or bio metabolites that can be obtained from it.

Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dental diseases are among the common health issues experienced around the world. Dental caries is one of the most predominant oral diseases worldwide. Major factors associated with caries development include poor oral hygiene, the content of specific carbohydrates in the diet, dental biofilm formation, the cariogenic microbial load, reduction in salivary flow, insufficient fluoride exposure, gingival recession, genetic factors, and lack of personal attention to one's dental health. Several preventive measures have been implemented to reduce the risk of the development of caries. Probiotics are live microbes that when administered in suitable amounts confer health benefits on the host; they are recognized as potential adjunct therapeutic agents for several diseases. The present manuscript summarizes recent findings on the role of probiotics in dental caries prevention and the possible mechanisms of probiotic effects. Review of the literature indicates the regular consumption of probiotic products significantly reduced the risk of caries by inhibiting cariogenic bacteria and enriching commensal microbes in the oral cavity. Buffering the salivary pH, production of bacteriocin and enzymes (dextranase, mutanase, and urease), the capacity of competing for the adhesion and colonization on tooth surfaces are the possible mechanisms behind the beneficial effect of probiotics. Further studies are necessary to address the efficacy of long-term probiotic supplementation on the control of dental diseases and the influence of childhood probiotic supplementation on the risk of caries development.

L-asparaginases (L-ASNases; EC 3.5.1.1) are aminohydrolases that catalyze the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid and ammonia, resulting in the death of acute lymphoblastic leukemic cells and other blood cancer cells. In this study, Bacillus sonorensis (accession number MK523484) uncharacterized L-ASNase gene (accession number MN562875) was isolated by polymerase chain reaction (PCR), cloned into pET28a (+) vector, and expressed in Escherichia coli as a cytosolic protein. The recombinant enzyme was purified by affinity chromatography at 23.79-fold and 49.37% recovery. Denaturing polyacrylamide gel (10%) analysis of the purified enzyme resulted in a single protein band at 36 kDa that immunoreacted strongly with 6His-tag monoclonal antibody. The purified enzyme exhibited optimal activity at 45 °C and pH 7.0 and retained 92% and 85% of its initial activity after incubation for 60 min at 37 °C and 45 °C, respectively. The purified enzyme exhibited substrate specificity toward L-asparagine and low glutaminase activity (15.72%) toward L-glutamine at a concentration of 10 mM. The Km and Vmax values were 2.004 mM and 3723 µmol min 1- , respectively.

Infections caused by carbapenem-resistant Enterobacterales (CRE) present an important therapeutic problem, as there are limited number of effective therapeutic alternatives available. In this study, phenotypic and genotypic methods were used to characterize carbapenemase-production and other resistance-determinants (AmpC and ESBL-production, efflux pump-overexpression) in 50 isolates (Klebsiella spp. n = 35, Escherichia coli n = 12 and Enterobacter cloacae complex n = 3) collected at the Albert Szent-Györgyi Clinical Center (University of Szeged) between 2014 and 2017. Minimum inhibitory concentrations of meropenem, sulfamethoxazole/trimethoprim, tigecycline, amikacin, moxifloxacin, colistin and fosfomycin were also determined. 24% of isolates were AmpC-producers, while 30% carried blaCTX-M ESBL-genes. Carbapenemase-genes were detected in 18 (36%) of the tested isolates: in 2 isolates blaNDM, in 6 isolates blaOXA-48-like and in 12 isolates, blaVIM was detected by PCR. The species-distribution for isolates positive for carbapenemase-genes was the following: Klebsiella pneumoniae n = 11, Klebsiella oxytoca n = 1, E. coli n = 5, E. cloacae complex n = 1. Efflux pump-overexpression based on the PAβN-screening agar was shown in n = 3 of the tested strains. In nine isolates (18%), carbapenemase and ESBL-genes were detected simultaneously. Highest levels of resistance were noted for fosfomycin (74%) and moxifloxacin (70%), while all isolates were susceptible to colistin. Among applied phenotypic tests in this study the modified carbapenem inactivation method (mCIM) proved to be the most accurate one compared to that of PCR results.

Dibutyl phthalate (DBP) is an environmental pollutant that can threaten human health. The strain Arthrobacter sp. ZJUTW, isolated from the sludge of a river of Hangzhou, can efficiently degrade DBP. Its genomic and transcriptomic differences when cultivated with DBP compared with glucose revealed specific DBP metabolic pathways in the ZJUTW strain. The degrading gene clusters localize separately on a circular chromosome and a plasmid pQL1. Genes related to the initial steps of DBP degradation from DBP to phthalic acid (PA), the pehA gene, and pht gene cluster, are located on the plasmid pQL1. However, the pca gene cluster related to the transforming of intermediate protocatechuic acid (PCA) to acetyl-CoA, is located on the chromosome. After comparative analysis with the reported gene clusters, we found that there were a series of homologous genes in pht and pca gene clusters that contribute to the efficient degradation of DBP by ZJUTW. In addition, transcriptomic analysis suggested a synergistic effect between pht and pca clusters, which also favor ZJUTW allowing it to efficiently degrade DBP. Combined genomic and transcriptomic analyses revealed a complete DBP metabolic pathway in Arthrobacter sp. ZJUTW that is different from that of other reported Arthrobacter strains. After necessary modification based on its metabolic characteristics, Arthrobacter sp. ZJUTW or its derivatives might represent promising candidates for the bioremediation of DBP pollution., (© 2020 Wiley Periodicals LLC.)

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1 P443L ) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans , indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Maunders et al.)

Accumulation of stress ethylene in plants due to osmotic stress is a major challenge for the achievement of optimum sweet corn crop yield with limited availability of irrigation water. A significant increase in earth's temperature is also making the conditions more crucial regarding the availability of ample quantity of irrigation water for crops production. Plant growth promoting rhizobacteria (PGPR) can play an imperative role in this regard. Inoculation of rhizobacteria can provide resistance and adaptability to crops against osmotic stress. In addition, these rhizobacteria also have potential to solve future food security issues. That's why the current study was planned to examine the efficacious functioning of Pseudomonas fluorescens strains on yields and physiological characteristics of sweet corn (Zea mays L. var saccharata) under different levels of irrigation. Three irrigation levels i.e., 100% (I 100 no stress), 80% (I 80 ), and 60% (I 60 ) were used during sweet corn cultivation. However, there were four rhizobacteria strains i.e., P. fluorescens P 1 , P. fluorescens P 3 , P. fluorescens P 8 , P. fluorescens P 14 which were used in the experiment. The results showed that severe water stress (60% of plant water requirement) decreased chlorophyll a, chlorophyll b, and total chlorophyll contents, Fv/Fm ratio and nutrients uptake. A significant increase in F 0 , F m , proline, total soluble sugars, catalase (CAT) and peroxidase (POX) activity led to less ear yield and canned seed yield. Combination of four strains significantly increased the yield traits of sweet corn i.e., ear and (44%) and canned seed yield (27%) over control. The highest promoting effect was observed in the combination of four strains treatment and followed by P 1 strain in reducing the harmful effects of drought stress and improving sweet corn productivity. However, P 14 gave minimum improvement in growth and yield indices under limited availability of water. In conclusion, combination of four strains inoculation is an efficacious approach for the achievement of better yield of sweet corn under osmotic stress.

Extensively drug resistant Acinetobacter baumannii (XDR-Ab), has emerged as an important pathogen in several outbreaks. The aim of our study was to investigate the eventual genetic relatedness of XDR-Ab strains recovered from burn patients and environment sites in the largest Tunisian Burn Intensive Care Unit (BICU) and to characterize β-lactamase encoding genes in these strains. Between March 04th, 2019 and April 22nd, 2019 an outbreak of XDR-Ab was suspected. Environmental screening was done. All isolates were screened by simplex PCR for β-lactamase genes. Genetic relatedness was determined by pulsed field gel electrophoresis (PFGE) of ApaI-digested total DNA. During the study period, 21 strains of A. baumannii were isolated in burn patients, mainly in blood culture (n = 7) and central vascular catheter (n = 6). All strains were susceptible to colistin but resistant to imipenem (n = 23), ciprofloxacin (n = 23), amikacin (n = 22), tigecyclin (n = 5) and rifampicin (n = 4). The blaOXA-51-like, blaOXA23, and blaADC genes were present in all strains. These resistance determinants were associated with blaPER-1 in 10 strains. The ISAba1 was inserted upstream of blaOXA-23 in all isolates. PFGE revealed two major clusters A (n = 11) and B (n = 5). This is the first description in Tunisia of clonally related PER-1 producing XDR-Ab in burn patients with probable environmental origin.

Bacterial pneumonia is one of the most prevalent infectious diseases and has high mortality in sensitive patients (children, elderly and immunocompromised). Although an infection, the disease alters the alveolar epithelium homeostasis and hinders normal breathing, often with fatal consequences. A special case is hospitalized aged patients, which present a high risk of infection and death because of the community acquired version of the Streptococcus pneumoniae pneumonia. There is evidence that early antibiotics treatment decreases the inflammatory response during pneumonia. Here, we investigate mechanistically this strategy using a multi-level mathematical model, which describes the 24 first hours after infection of a single alveolus from the key signaling networks behind activation of the epithelium to the dynamics of the local immune response. With the model, we simulated pneumonia in aged and young patients subjected to different antibiotics timing. The results show that providing antibiotics to elderly patients 8 h in advance compared to young patients restores in aged individuals the effective response seen in young ones. This result suggests the use of early, probably prophylactic, antibiotics treatment in aged hospitalized people with high risk of pneumonia.