scholarly journals Functional divergence and potential mechanisms of the duplicate recA genes in Myxococcus xanthus

2019 ◽  
Author(s):  
Duo-hong Sheng ◽  
Yi-xue Wang ◽  
Miao Qiu ◽  
Jin-yi Zhao ◽  
Xin-jing Yue ◽  
...  
Keyword(s):  
Uv Irradiation ◽  
Functional Divergence ◽  
Myxococcus Xanthus ◽  
Dna Recombination ◽  
Dna Repeats ◽  
E Coli ◽  
Basal Expression ◽  
Basal Expression Level ◽  
Irradiation Resistance

AbstractRecA is a ubiquitous multifunctional protein for bacterial homologous recombination and SOS response activation. Myxococcus xanthus DK1622 possesses two recA genes, and their functions and mechanisms are almost unclear. Here, we showed that the transcription of recA1 (MXAN_1441) was less than one-tenth of recA2 (MXAN_1388). Expressions of the two recA genes were both induced by ultraviolet (UV) irradiation, but in different periods. Deletion of recA1 did not affect the growth, but significantly decreased the UV-irradiation survival, the homologous recombination ability, and the induction of the LexA-dependent SOS genes. Comparably, the deletion of recA2 markedly prolonged the lag phase for cellular growth and antioxidation of hydrogen peroxide, but did not change the UV-irradiation resistance and the SOS-gene inducibility. The two RecA proteins are both DNA-dependent ATPase enzymes. We demonstrated that RecA1, but not RecA2, had in vitro DNA recombination capacity and LexA-autolysis promotion activity. Transcriptomic analysis indicated that the duplicate RecA2 has evolved to mainly regulate the gene expressions for cellular transportation and antioxidation. We discuss the potential mechanisms for the functional divergence. This is the first time to clearly determine the divergent functions of duplicated recA genes in bacterial cells. The present results highlight that the functional divergence of RecA duplicates facilitates the exertion of multiple RecA functions.Author summaryMyxobacteria has a large-size genome, contains many DNA repeats that are rare in the prokaryotic genome. It encodes bacterial RecA that could promote recombination between homologous DNA sequences. How myxobacteria avoid the undesired recombination between DNA repeats in its genome is an interesting question. M. xanthus encodes two RecA proteins, RecA1 (MXAN_1441) and RecA2 (MXAN_1388). Both RecA1 and RecA2 shows more than 60% sequence consistency with E. coli RecA (EcRecA) and can partly restore the UV resistance of E. coli recA mutant. Here, our results proved their divergent functions of the two RecAs. RecA1 retains the ability to catalyze DNA recombination, but its basal expression level is very low. RecA2 basal expression level is high, but no recombination activity is detected in vitro. This may be a strategy for M. xanthus to adapt to more repetitive sequences in its genome and avoid incorrect recombination.HighlightsM. xanthus has two recAs, which are expressed with significantly different levels. Both recAs are inducible by UV irradiation, but in different stages.The absence of recA1 reduces bacterial UV-irradiation resistance, while the absence of recA2 delays bacterial growth and antioxidant capacity.RecA1 retains the DNA recombination and SOS induction abilities, while RecA2 has evolved to regulate the expression of genes for cellular transport and antioxidation.

scholarly journals Role of Hfq in Genome Evolution: Instability of G-Quadruplex Sequences in E. coli

2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Virali J. Parekh ◽  
Brittany A. Niccum ◽  
Rachna Shah ◽  
Marisa A. Rivera ◽  
Mark J. Novak ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Dna Repeats ◽  
Loop Formation ◽  
Bacterial Gene ◽  
Quadruplex Dna ◽  
E Coli ◽  
Alternative Structures ◽  
Bacterial Gene Expression ◽  
G Quadruplex

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

STUDIES ON MECHANISM OF REPAIR OF ULTRAVIOLET-IRRADIATED VIRAL AND BACTERIAL DNA IN VIVO AND IN VITRO

1965 ◽  
Vol 43 (7) ◽  
pp. 1207-1219 ◽  
Author(s):  
Emanuel Riklis
Keyword(s):  
Uv Irradiation ◽  
Soluble Fraction ◽  
Cell Extract ◽  
Host Cells ◽  
Bacterial Dna ◽  
E Coli ◽  
Thymine Dimers ◽  
K 12

The formation of thymine dimers [Formula: see text] from adjacent intrastrand thymines by ultraviolet (UV) irradiation in DNA was studied under different conditions. When thymine-2-C14 DNA was exposed in quartz tubes to 0.5 × 106 ergs/mm2 ultraviolet irradiation, two photoproducts were formed: "a" + [Formula: see text]. The concentration formed in dry DNA was only about [Formula: see text] of that formed in wet DNA.The survival of T1 phage in the dark in the resistant (uvr+) and the sensitive (uvr−) mutants of E. coli K-12 after UV irradiation of the phage in the wet and dry state is markedly dependent on the state of the phage during irradiation. Survival of T1 phage, when UV irradiated dry, was the same in the sensitive as in the resistant host cells, over a wide range of UV doses, while a marked difference in sensitivity existed when it was UV irradiated wet. Similar survival was obtained also by photoreactivation. These results correspond with the notion that thymine dimers are involved both in photoreactivation and in dark (host cell) reactivation.Thymine-requiring E. coli K-12 cells were mutated to a radioresistant strain uvr+ (AB 2416) and a radiosensitive strain uvr− (AB 2419).Irradiation of cells with a dose of 1000 ergs/mm2, followed by incubation of the cells in the dark in enriched M9 media, stopped by addition of 5% trichloroacetic acid, hydrolysis of the acid-insoluble fraction and the acid-soluble fraction in trifluoroacetic acid at 175 °C, and separation of the products by paper chromatography, showed that the two irradiation products "a" + [Formula: see text], which were formed in the bacterial DNA, are excised from the DNA in the uvr+ strain and appear in the acid-soluble fraction. No such excision occurred upon incubation of the radiosensitive strain uvr−.Incubation of the cells under light showed that photoreactivation prevails in the radiosensitive strain, i.e. disappearance of "a" + [Formula: see text] from the DNA, without their appearance in the acid-soluble fraction, while dark reactivation prevailed in the uvr+ strain, a result that indicates a stronger affinity of the dark reactivating system to UV-irradiated DNA. Cell extracts prepared by breaking the cells in a French press in Tris buffer, pH 7.5, plus 10−3 M Mg++ plus 10−3 M mercapto-ethanol showed a similar mechanism; the two irradiation products were excised from DNA by a uvr+ cell extract and not by a uvr− cell extract. Extract of uvr+ cells brought about excision of photoproducts from DNA of the uvr− cell extract.The results suggest that an enzyme, capable of excising thymine dimers, is present in the radioresistant cell as part of the system of repair of DNA from UV irradiation, and its mechanism is demonstrated, both in vivo and in vitro.

scholarly journals Photoreactivation of Escherichia coli after Low- or Medium-Pressure UV Disinfection Determined by an Endonuclease Sensitive Site Assay

2002 ◽  
Vol 68 (12) ◽  
pp. 6029-6035 ◽  
Author(s):  
Kumiko Oguma ◽  
Hiroyuki Katayama ◽  
Shinichiro Ohgaki
Keyword(s):  
Escherichia Coli ◽  
Uv Irradiation ◽  
Genomic Dna ◽  
Uv Disinfection ◽  
E Coli ◽  
Medium Pressure ◽  
Pyrimidine Dimers ◽  
Sensitive Site ◽  
Uv Lamp

ABSTRACT Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E. coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E. coli. In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of E. coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Антимикробная активность лиофилизированного водного извлечения Caragana jubata (Pall.) Poir.

2020 ◽  
Vol 54 (3) ◽  
pp. 42-44
Author(s):  
Павел Алексеевич Какорин ◽  
Татьяна Владимировна Фатеева ◽  
Ольга Ивановна Терешкина ◽  
Ирина Борисовна Перова ◽  
Галина Владиславовна Раменская ◽  
...  
Keyword(s):  
E Coli

На основании ранее проведенных исследований установлен профиль флавоноидов лиофилизированного водного извлечения, полученного из побегов C. jubata. В связи с тем, что, согласно данным литературы, флавоноиды являются потенциальными ингибиторами микроорганизмов, проведено изучение антимикробной активности лиофилизата в опытах in vitro с использованием скринигового метода определения антимикробной активности для препаратов растительного происхождения. При изучении бактериостатической и фунгистатической активности в опытах in vitro использовали метод двукратного серийного разведения препаратов в жидких питательных средах. В результате исследования лиофилизированного водного извлечения караганы гривастой установлено наличие умеренной антимикробной активности в отношении всех изученных штаммов патогенных микроорганизмов: грамположительных и грамотрицательных бактерий (S. aureus, E. coli, P. vulgaris, P. aeruginosa), дрожжеподобных и мицелиальных грибов (C. albicans, M. canis). Полученные данные позволяют рекомендовать лиофилизированное водное извлечение караганы гривастой для создания на его основе лекарственных форм наружного применения для лечения заболеваний кожи и слизистых оболочек, связанных с бактериальным воспалительным процессом.

Влияние in vitro изолированного и сочетанного с антибактериальными средствами применения бовгиалуронидазы азоксимер на целостность бактериальной биопленки и жизнеспосоность микроорганизмов

2020 ◽  
Vol 83 (2) ◽  
pp. 38-44
Author(s):  
Е. Ю. Тризна ◽  
Д. Р. Байдамшина ◽  
Александр А. Виницкий ◽  
А. Р. Каюмов
Keyword(s):  
E Coli

Исследована способность лиофилизата бовгиалуронидазы азоксимера («Лонгидаза») разрушать бактериальные биопленки S. aureus, E. faecalis, E. coli, а также сочетанное действие препарата с антибактериальными средствами. Показано, что 2 ч инкубации бовгиалуронидазы азоксимер в концентрации 750 – 1500 МЕ/мл вызывает двукратное снижение биомассы матрикса зрелых биопленок E. faecalis и E. coli, и на 60 % — S. aureus. Данный ферментный препарат не влияет на образование бактериальных биопленок. При сочетанном применении с антибактериальными средствами препарат повышает их эффективность в отношении бактерий в составе биопленок. Так, концентрация ципро-флоксацина и амоксициллина, необходимая для снижения количества КОЕ на 3 порядка в биопленке E. faecalis, в присутствии бовгиалуронидазы азоксимера снижается в 16 раз (p < 0,05). В присутствии фермента в 16 раз меньшие концентрации цефуроксима, фосфомицина, ципрофлоксацина и амикацина достаточны для снижения количества КОЕ на 3 порядка в биопленке E. coli (p < 0,05), и в значительно меньшей концентрации цефуроксим оказывает бактерицидное действие на клетки в биопленке S. aureus (p < 0,05). Вероятно, бовгиалуронидаза азоксимер увеличивает проникновение антибактериальных средств к клеткам бактерий в биопленке, что обеспечивает потенцирование их антибактериального эффекта. Такое действие ферментного препарата позволяет снизить дозу и повысить безопасность антибактериальных средств при сохранении их эффективности.

Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

2019 ◽  
Vol 35 (6) ◽  
pp. 91-101
Author(s):  
F.A. Klebanov ◽  
S.E. Cheperegin ◽  
D.G. Kozlov
Keyword(s):  
Penicillium Chrysogenum ◽  
Protein Denaturation ◽  
Biologically Active ◽  
Cultivation Temperature ◽  
Target Proteins ◽  
E Coli ◽  
Complete Inactivation ◽  
Target Molecules ◽  
Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

In Vitro Holdase Activity of E. coli Small Heat-Shock Proteins IbpA, IbpB and IbpAB: A Biophysical Study with Some Unconventional Techniques

2014 ◽  
Vol 21 (6) ◽  
pp. 564-571 ◽  
Author(s):  
Sourav Roy ◽  
Monobesh Patra ◽  
Suman Nandy ◽  
Milon Banik ◽  
Rakhi Dasgupta ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Small Heat Shock Proteins ◽  
E Coli ◽  
Biophysical Study ◽  
Shock Proteins

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

Acetate Kinase (AcK) is Essential for Microbial Growth and Betel-derived Compounds Potentially Target AcK, PhoP and MDR Proteins in M. tuberculosis, V. cholerae and Pathogenic E. coli: An in silico and in vitro Study

2019 ◽  
Vol 18 (31) ◽  
pp. 2731-2740 ◽  
Author(s):  
Sandeep Tiwari ◽  
Debmalya Barh ◽  
M. Imchen ◽  
Eswar Rao ◽  
Ranjith K. Kumavath ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
In Silico ◽  
Pathogenic Bacteria ◽  
In Vitro Study ◽  
Docking Studies ◽  
Acetate Kinase ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Novel Target

Background: Mycobacterium tuberculosis, Vibrio cholerae, and pathogenic Escherichia coli are global concerns for public health. The emergence of multi-drug resistant (MDR) strains of these pathogens is creating additional challenges in controlling infections caused by these deadly bacteria. Recently, we reported that Acetate kinase (AcK) could be a broad-spectrum novel target in several bacteria including these pathogens. Methods: Here, using in silico and in vitro approaches we show that (i) AcK is an essential protein in pathogenic bacteria; (ii) natural compounds Chlorogenic acid and Pinoresinol from Piper betel and Piperidine derivative compound 6-oxopiperidine-3-carboxylic acid inhibit the growth of pathogenic E. coli and M. tuberculosis by targeting AcK with equal or higher efficacy than the currently used antibiotics; (iii) molecular modeling and docking studies show interactions between inhibitors and AcK that correlate with the experimental results; (iv) these compounds are highly effective even on MDR strains of these pathogens; (v) further, the compounds may also target bacterial two-component system proteins that help bacteria in expressing the genes related to drug resistance and virulence; and (vi) finally, all the tested compounds are predicted to have drug-like properties. Results and Conclusion: Suggesting that, these Piper betel derived compounds may be further tested for developing a novel class of broad-spectrum drugs against various common and MDR pathogens.

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