scholarly journals Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector

2006 ◽  
Vol 72 (1) ◽  
pp. 527-535 ◽  
Author(s):  
Ju-Hoon Lee ◽  
Daniel J. O’Sullivan
Keyword(s):  
Recombination Event ◽  
Sequence Similarity ◽  
Bifidobacterium Longum ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Rhodococcus Rhodochrous ◽  
Gram Positive ◽  
High Sequence Identity ◽  
Chloramphenicol Resistance ◽  
E Coli

ABSTRACT Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

scholarly journals Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

2007 ◽  
Vol 74 (5) ◽  
pp. 1385-1393 ◽  
Author(s):  
Torsten Gursinsky ◽  
Daniel Gröbe ◽  
Angelika Schierhorn ◽  
Jana Jäger ◽  
Jan R. Andreesen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Insertion Sequence ◽  
Sequence Similarity ◽  
Elongation Factor ◽  
Gram Positive ◽  
Secis Element ◽  
E Coli ◽  
Uga Codon ◽  
Selenophosphate Synthetase ◽  
Sequence Requirements

ABSTRACT Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of 75Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNASec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of 75Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.

Structure-Activity Relationship and Antimicrobial Evaluation of N-Phenylpyrazole Curcumin Derivatives

2020 ◽  
Vol 16 (4) ◽  
pp. 481-488
Author(s):  
Heli Sanghvi ◽  
Satyendra Mishra
Keyword(s):  
Antibacterial Activity ◽  
Gram Negative Bacteria ◽  
Pyrazole Derivatives ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Curcumin Derivatives ◽  
Structure Activity ◽  
Electron Donating Groups ◽  
Derivatives Of

Background: Curcumin, one of the most important pharmacologically significant natural products, has gained significant consideration among scientists for decades since its multipharmacological activities. 1, 3-Dicarbonyl moiety of curcumin was found to be accountable for the rapid degradation of curcumin molecule. The aim of present work is to replace 1, 3-dicarbonyl moiety of curcumin by pyrazole and phenylpyrazole derivatives with a view to improving its stability and to investigate the role of substitution in N-phenylpyrazole curcumin on its antibacterial activity against both Gram-positive as well as Gram-negative bacteria. Methods: Pyrazole derivatives of curcumin were prepared by heating curcumin with phenyhydrazine/ substituted phenyhydrazine derivatives in AcOH. The residue was purified by silica gel column chromatography. Structures of purified compounds were confirmed by 1H NMR and Mass spectroscopy. The synthesized compounds were evaluated for their antibacterial activity by the microdilution broth susceptibility test method against gram positive (S. aureus) and gram negative (E. coli). Results: Effects of substitution in N-phenylpyrazole curcumin derivatives against S. aureus and E. coli were studied. The most active N-(3-Nitrophenylpyrazole) curcumin (12) exhibits twenty-fold more potency against S. aureus (MIC: 10μg/mL)) and N-(2-Fluoroophenylpyrazole) curcumin (5) fivefold more potency against E. coli (MIC; 50 μg/mL) than N-phenylpyrazole curcumin (4). Whereas, a remarkable decline in anti-bacterial activity against S. aureus and E. coli was observed when electron donating groups were incorporated in N-phenylpyrazole curcumin (4). Comparative studies of synthesized compounds suggest the effects of electron withdrawing and electron donating groups on unsubstituted phenylpyrazole curcumin (4). Conclusion: The structure-activity relationship (SAR) results indicated that the electron withdrawing and electron donating at N-phenylpyrazole curcumin played key roles for their bacterial inhibitory effects. The results of the antibacterial evaluation showed that the synthesized pyrazole derivatives of curcumin displayed moderate to very high activity in S. aureus. In conclusion, the series of novel curcumin derivatives were designed, synthesized and tested for their antibacterial activities against S. aureus and E. coli. Among them, N-(3-Nitrophenylpyrazole curcumin; 12) was most active against S. aureus (Gram-positive) and N-(2-Fluoroophenylpyrazole) curcumin (5) against E. coli (Gram-negative) bacteria.

scholarly journals A novel bis(pyrazolyl)methane compound as a potential agent against Gram-positive bacteria

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Pedro Seguí ◽  
John J. Aguilera-Correa ◽  
Elena Domínguez-Jurado ◽  
Christian M. Sánchez-López ◽  
Ramón Pérez-Tanoira ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Inhibitory Effect ◽  
Eukaryotic Cell ◽  
Liver Carcinoma ◽  
Gram Positive ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Therapeutic Compounds ◽  
Toxic Concentrations

AbstractThis study was designed to propose alternative therapeutic compounds to fight against bacterial pathogens. Thus, a library of nitrogen-based compounds bis(triazolyl)methane (1T–7T) and bis(pyrazolyl)methane (1P–11P) was synthesised following previously reported methodologies and their antibacterial activity was tested using the collection strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. Moreover, the novel compound 2P was fully characterized by IR, UV–Vis and NMR spectroscopy. To evaluate antibacterial activity, minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs), minimum biofilm inhibitory concentrations (MBICs), and minimum biofilm eradication concentrations (MBECs) assays were carried out at different concentrations (2–2000 µg/mL). The MTT assay and Resazurin viability assays were performed in both human liver carcinoma HepG2 and human colorectal adenocarcinoma Caco-2 cell lines at 48 h. Of all the synthesised compounds, 2P had an inhibitory effect on Gram-positive strains, especially against S. aureus. The MIC and MBC of 2P were 62.5 and 2000 µg/mL against S. aureus, and 250 and 2000 µg/mL against E. faecalis, respectively. However, these values were > 2000 µg/mL against E. coli and P. aeruginosa. In addition, the MBICs and MBECs of 2P against S. aureus were 125 and > 2000 µg/mL, respectively, whereas these values were > 2000 µg/mL against E. faecalis, E. coli, and P. aeruginosa. On the other hand, concentrations up to 250 µg/mL of 2P were non-toxic doses for eukaryotic cell cultures. Thus, according to the obtained results, the 2P nitrogen-based compound showed a promising anti-Gram-positive effect (especially against S. aureus) both on planktonic state and biofilm, at non-toxic concentrations.

scholarly journals Asymptomatic Bacteriuria among Pregnant Women Attending Tertiary Care Hospital in Lucknow, India

2021 ◽  
pp. 1-8
Author(s):  
Naimshree Sonkar ◽  
Malay Banerjee ◽  
Suman Gupta ◽  
Absar Ahmad
Keyword(s):  
Haemoglobin Level ◽  
Pregnant Women ◽  
Antimicrobial Susceptibility ◽  
Tertiary Care Hospital ◽  
Tertiary Care ◽  
Asymptomatic Bacteriuria ◽  
Care Hospital ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli

Introduction: Asymptomatic bacteriuria (ASB) is the presence of actively multiplying bacteria within the urinary tract with absence of any symptoms, resulting in adverse pregnancy outcomes. This research study was done in order to review prevalence, antimicrobial susceptibility profile, and factors associated with ASB occurring in female patients who are pregnant and being treated at a tertiary care hospital in Lucknow, India. Method and Materials: This is a cross-sectional study done among 216 pregnant women attending a hospital for antenatal check-ups. Clean catch midstream urine samples were collected and examined microscopically, and semi-quantitative culture was done on blood agar and MacConkey agar. Isolates were identified by colony morphology and biochemical tests, and antimicrobial susceptibility testing was done by using the Kirby-Bauer method. Results: Of the 216 pregnant women, 36 (16.7%) tested positive for ASB. The female gestational period, haemoglobin level, and BMI were significantly associated with ASB. Logistic regression also showed that higher haemoglobin level was less likely to ASB (AOR = 0.42, 95% confidence interval: 0.202–0.88, p = 0.021). The predominant and usual isolates were E. coli (n = 22, 61.1%), followed by Cons (n = 6, 16.7%), and S. aureus (3, 8.3%). All Gram-negative isolates were mostly sensitive to most of the drugs like piperacillin-tazobactam, cefepime, nitrofurantoin, and meropenem but were 100% resistant to ampicillin. Similarly, Gram-positive isolates were sensitive to ampicillin, vancomycin, linezolid, and nitrofurantoin but 100% resistant to co-trimoxazole. Conclusion: The present study shows the existence of ASB was 16.7% among women who are pregnant. Pregnancy duration, haemoglobin level, and BMI were significantly associated with ASB. The isolates identified more frequently were E. coli (61.16%), Cons (16.7%), and S. aureus (8.3%). All isolates which were Gram-negative were mostly sensitive to most of the drugs but were 100% resistant to ampicillin. Similarly, Gram-positive isolates were sensitive to most of the drugs but 100% resistant to co-trimoxazole.

scholarly journals Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Naomi Shimokawa-Chiba ◽  
Claudia Müller ◽  
Keigo Fujiwara ◽  
Bertrand Beckert ◽  
Koreaki Ito ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stop Codon ◽  
Release Factor ◽  
Positive Bacterium ◽  
Gram Positive ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Dead End ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus

AbstractRescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

Preparation of bioactive and antibacterial PMMA-based bone cement by modification with quaternary ammonium and alkoxysilane

2021 ◽  
pp. 088532822110044
Author(s):  
Haiyang Wang ◽  
Toshinari Maeda ◽  
Toshiki Miyazaki
Keyword(s):  
Antibacterial Activity ◽  
Methyl Methacrylate ◽  
Bone Cement ◽  
Setting Time ◽  
Antibacterial Properties ◽  
The Body ◽  
Apatite Formation ◽  
Gram Positive ◽  
E Coli ◽  
Gram Positive Bacteria

Bone cement based on poly(methyl methacrylate) (PMMA) powder and methyl methacrylate (MMA) liquid is a very popular biomaterial used for the fixation of artificial joints. However, there is a risk of this cement loosening from bone because of a lack of bone-bonding bioactivity. Apatite formation in the body environment is a prerequisite for cement bioactivity. Additionally, suppression of infection during implantation is required for bone cements to be successfully introduced into the human body. In this study, we modified PMMA cement with γ-methacryloxypropyltrimetoxysilane and calcium acetate to introduce bioactive properties and 2-( tert-butylamino)ethyl methacrylate (TBAEMA) to provide antibacterial properties. The long-term antibacterial activity is attributed to the copolymerization of TBAEMA and MMA. As the TBAEMA content increased, the setting time increased and the compressive strength decreased. After soaking in simulated body fluid, an apatite layer was detected within 7 days, irrespective of the TBAEMA content. The cement showed better antibacterial activity against Gram-negative E. Coli than Gram-positive bacteria; however, of the Gram-positive bacteria investigated, B. subtilis was more susceptible than S. aureus.

scholarly journals 1699. Variation of antimicrobial resistance by age groups for outpatient UTI isolates in US females: A multicenter evaluation from 2011 to 2019

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S832-S832
Author(s):  
Keith S Kaye ◽  
Vikas Gupta ◽  
Aruni Mulgirigama ◽  
Ashish V Joshi ◽  
Nicole Scangarella-Oman ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Age Groups ◽  
Outpatient Setting ◽  
Research Database ◽  
Age Group ◽  
Becton Dickinson ◽  
Gram Positive ◽  
Multicenter Cohort Study ◽  
Material Support ◽  
E Coli

Abstract Background An estimated 12% of women experience ≥ 1 episode of urinary tract infection (UTI) annually. Incidence is bimodal, with peaks occurring in young, sexually active women (18–24 years) and in post-menopausal women. Previous studies suggest the prevalence of antimicrobial resistance (AMR) in UTI is rising; however recent AMR data for community-acquired UTI are lacking. We estimated the prevalence of AMR among US females with outpatient UTI in 2011–2019, stratified by age. Methods A retrospective, multicenter, cohort study of AMR among non-duplicate urine isolates in US females (≥ 12 years of age) from 296 institutions from 2011–2019 (BD Insights Research Database, Franklin Lakes, NJ). Phenotypes examined for Enterobacterales (ENT) were: extended spectrum β-lactamase positive (ESBL+; determined by commercial panels or intermediate/resistant to ceftriaxone, cefotaxime, ceftazidime or cefepime); nitrofurantoin (NFT) not-susceptible (NS); fluoroquinolone (FQ) NS; trimethoprim-sulfamethoxazole (TMP-SMX) NS; and NS to ≥ 2 or ≥ 3 drug classes (including ESBL+). Gram-positive phenotypes were, methicillin resistant S. aureus and S. saprophyticus and vancomycin-resistant Enterococcus. Isolates were stratified by patient age (≥ 12 to < 18, ≥ 18 to < 55, ≥ 55 to < 65, ≥ 65 to < 75, ≥ 75 years). Chi-square tests were used to evaluate AMR difference between groups. Results In total, urine isolates were collected from 106 to 296 (2011–2019) US sites. Overall, the prevalence of antimicrobial NS increased with age for all E. coli phenotypes (all P< 0.001; Table 1), and for non-E. coli ENT (all P< 0.001), except NFT NS, which decreased from 70.6% to 59.7% (P=0.002; Table 2). The greatest difference between age groups in prevalence of resistance was observed for FQ NS E.coli: 5.8% (≥ 12 to < 18 years) vs 34.5% (≥ 75 years). For the multi-drug resistant E. coli phenotypes, resistance increased with age, ranging from 4.8–22.4% and 0.9–6.5% for ≥ 2 and ≥ 3 drug NS, respectively. Overall, the prevalence of resistance for Gram-positive phenotypes increased with age (all P< 0.001; Table 3). Table 1. Prevalence of antimicrobial resistance among E. coli isolates in US females with outpatient UTI by age group. Table 2. Prevalence of antimicrobial resistance among non-E. coli ENT isolates in US females with outpatient UTI by age group. Table 3. Prevalence of antimicrobial resistance among Gram-positive isolates in US females with outpatient UTI by age group. Conclusion The prevalence of AMR in E. coli and non-E. coli ENT increased with age among US females presenting for care in the outpatient setting overall. A similar trend increase by age is also seen in Gram-positive isolates. Disclosures Vikas Gupta, PharmD, BCPS, Becton, Dickinson and Company (Employee, Shareholder)GlaxoSmithKline plc. (Other Financial or Material Support, Funding) Aruni Mulgirigama, MBBS, GlaxoSmithKline plc. (Employee, Shareholder) Ashish V. Joshi, PhD, GlaxoSmithKline plc. (Employee, Shareholder) Nicole Scangarella-Oman, MS, GlaxoSmithKline plc. (Employee, Shareholder) Kalvin Yu, MD, Becton, Dickinson and Company (Employee)GlaxoSmithKline plc. (Other Financial or Material Support, Funding) Gang Ye, PhD, Becton, Dickinson and Company (Employee)GlaxoSmithKline plc. (Other Financial or Material Support, Funding) Fanny S. Mitrani-Gold, MPH, GlaxoSmithKline plc. (Employee, Shareholder)

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

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