Molecular analysis of mutatedLactobacillus acidophiluspromoter-like sequence P15

2000 ◽  
Vol 46 (10) ◽  
pp. 938-945 ◽  
Author(s):  
Slavica Arsenijevic ◽  
Ljubisa Topisirovic
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Molecular Analysis ◽  
Lactobacillus Acidophilus ◽  
Promoter Activity ◽  
Lactobacillus Reuteri ◽  
Promoter Sequence ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Promoter Probe

The promoter-like sequence P15 that was previously cloned from the chromosome of Lactobacillus acidophilus ATCC 4356 is active in Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus acidophilus, and Escherichia coli, but not in Lactococcus lactis. N-methyl-N-nitroso-N-guanidine (MNNG) mutagenesis of P15 was used to select for a promoter active in L. lactis MG1363. Molecular analysis of the mutated promoter (designated P16) revealed a 90 bp deletion and a T[Formula: see text]A transversion. This deletion, in combination with the addition to the transversion, created a promoter with putative -35 and -10 hexamers identical to the consensus promoter sequence found in E. coli and Bacillus subtilis vegetative promoters. The activity of P16 was measured by its ability to promote chloramphenicol resistance in different bacteria when inserted in the promoter-probe plasmid pBV5030 (designated pLA16). The MIC of chloramphenicol in L. lactis, L. reuteri, L. plantarum, E. coli, and L. acidophilus harbouring pLA16 were 30, 170, 180, >500, and 3 µg/mL, respectively. This represents an increase in promoter activity compared to P15 in L. reuteri of 3-fold, in L. plantarum of 9-fold, and in E. coli of at least 2.5-fold, but a decrease in L. acidophilus of 7-fold.Key words: Lactobacillus acidophilus, promoter-like sequence, mutagenesis.

Cloning and molecular analysis of promoter-like sequences isolated from the chromosomal DNA ofLactobacillus acidophilusATCC 4356

1997 ◽  
Vol 43 (1) ◽  
pp. 61-69 ◽  
Author(s):  
G. Djordjevic ◽  
B. Bojovic ◽  
N. Miladinov ◽  
L. Topisirovic
Keyword(s):  
Lactococcus Lactis ◽  
Molecular Analysis ◽  
Lactobacillus Acidophilus ◽  
Promoter Activity ◽  
Lactobacillus Reuteri ◽  
Bacterial Species ◽  
Chromosomal Dna ◽  
Complementary Dna ◽  
E Coli ◽  
Dna Strand

Promoter-like sequences from the chromosomal DNA of thermophilic strain Lactobacillus acidophilus ATCC 4356 were cloned. Analysis of the three DNA fragments showing promoter activity, designated P3, P6, and P15, were performed in Lactobacillus reuteri, Lactococcus lactis, and E. coli. The reporter cat-86 gene was expressed in all three bacterial species under control of the fragments P3 and P6. Fragment P15 showed promoter activity only in Lactobacillus reuteri and E. coli but not in Lactococcus lactis. The three host-specific transcriptional start points (TSPs) were used when transcription of the cat-86 gene was controlled by fragment P3 in Lactobacillus reuteri, E. coli, and Lactococcus lactis. Similarly, fragment P15 initiated transcription of the cat-86 gene at two distinctive sites in Lactobacillus reuteri and E. coli. Only within fragment P6, a common TSP was used in Lactobacillus reuteri and E. coli, but different from that used in Lactococcus lactis. Each TSP was preceded by the putative −35 and −10 hexamers. Computer analysis of the fragment P3 sequence revealed the existence of divergent promoterlike sequence (P3rev) located on the complementary DNA strand. Fragments P6 and P15 were also functional in Lactobacillus acidophilus ATCC 4356 from which chromosomal DNA they were originally cloned.Key words: Lactobacillus acidophilus, promoter-like sequences, regulation.

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.

Peptide nucleic acid restores colistin susceptibility through modulation of MCR-1 expression in Escherichia coli

2020 ◽  
Author(s):  
Xiaoming Wang ◽  
Yao Wang ◽  
Zhuoren Ling ◽  
Chaoyang Zhang ◽  
Mingming Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peptide Nucleic Acid ◽  
Promoter Activity ◽  
Sequence Variation ◽  
Promoter Sequence ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Expression Host ◽  
Antisense Approach ◽  
Increased Susceptibility

Abstract Background Plasmid-mediated mechanisms of drug resistance accelerate the spread of polymyxin resistance, leaving clinicians with few or no antibacterial options for the treatment of infections caused by MDR bacteria, especially carbapenemase-producing strains. Objectives To evaluate the associations among promoter sequence variation, mcr-1 expression, host factors and levels of colistin resistance and to propose antisense agents such as peptide nucleic acids (PNAs) targeting mcr-1 as a tool to restore colistin susceptibility through modulation of MCR-1 expression in Escherichia coli. Methods A β-galactosidase assay was performed to study mcr-1 promoter activity. Quantitative real-time PCR and western blot assays were used to identify the expression level of MCR-1 in WT strains and transformants. Three PNAs targeting different regions of mcr-1 were designed and synthesized to determine whether they can effectively inhibit MCR-1 expression. MIC was measured to test colistin susceptibility in the presence or absence of PNA-1 in mcr-1-carrying E. coli. Results Variation in the mcr-1 promoter sequence and host species affect promoter activity, MCR-1 expression levels and colistin MICs. One PNA targeting the ribosome-binding site fully inhibited the expression of mcr-1 at a concentration of 4 μM, resulting in significantly increased susceptibility to colistin. The MIC90 of colistin decreased from 8 to 2 mg/L (P < 0.05) in the presence of 4 μM PNA. Conclusions These findings suggest that the antisense approach is a possible strategy to combat mcr-1-mediated resistance as well as other causes of emerging global resistance.

Mixed culture models for predicting intestinal microbial interactions between Escherichia coli and Lactobacillus in the presence of probiotic Bacillus subtilis

2015 ◽  
Vol 6 (6) ◽  
pp. 871-877 ◽  
Author(s):  
J.J. Yang ◽  
C.C. Niu ◽  
X.H. Guo
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Mixed Culture ◽  
Lactobacillus Reuteri ◽  
Microbial Interactions ◽  
Lactobacillus Species ◽  
E Coli ◽  
Culture Models ◽  
Static Conditions

Bacillus has been proposed as a probiotic due to its in vivo effectiveness in the gastrointestinal tract through antimicrobial activities. The present study investigates the effects of Lactobacillus alone or in the presence of Bacillus subtilis MA139 on the inhibition of pathogenic Escherichia coli K88. Mixed cultures were used to predict the possible interactions among these bacteria within the intestinal tract of animals. B. subtilis MA139 was first assayed for its inhibition against E. coli K88 both under shaking and static culture conditions. A co-culture assay was employed under static conditions to test the inhibitory effects of Lactobacillus reuteri on E. coli K88, with or without addition of B. subtilis MA139. The results showed that B. subtilis MA139 had marked inhibition against E. coli K88 under shaking conditions and weak inhibition under static conditions. Lactobacillus alone as well as in combination with B. subtilis MA139 spores exerted strong inhibition against E. coli K88 under static conditions. However, the inhibition by Lactobacillus in combination with B. subilis spores was much higher than that by Lactobacillus alone (P<0.01). B. subtilis MA139 significantly decreased the pH and oxidation-reduction potential values of the co-culture broth compared to that of Lactobacillus alone (P<0.05). The viability of Lactobacillus increased when co-cultured with B. subtilis MA139 because of significantly higher Lactobacillus counts and lower pH values in the broth (P<0.05). The role of Bacillus in the mixed culture models suggests that Bacillus may produce beneficial effects by increasing the viability of lactobacilli and subsequently inhibiting the growth of pathogenic E. coli. Therefore, the combination of Bacillus and Lactobacillus species as a probiotic is recommended.

scholarly journals Evaluation of Bean and Soy Tempeh Influence on Intestinal Bacteria and Estimation of Antibacterial Properties of Bean Tempeh

2013 ◽  
Vol 62 (2) ◽  
pp. 189-194 ◽  
Author(s):  
MACIEJ KULIGOWSKI ◽  
IWONA JASIŃSKA-KULIGOWSKA ◽  
JACEK NOWAK
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Lactobacillus Acidophilus ◽  
Intestinal Microflora ◽  
Antibacterial Properties ◽  
Intestinal Bacteria ◽  
E Coli ◽  
Genus Lactobacillus ◽  
Human Digestive Tract ◽  
Growth Of Bacteria

In this study the effect of bean tempeh on the growth of Bacillus subtilis, Escherichia coli, Lactobacillus acidophilus and Lactobacillus paracasei bacteria was investigated. Antibacterial activity was observed only in relation to the bacteria Bacillus subtilis. The effect of tempeh products on human intestinal microflora was also assessed. Bean and soy tempeh were culinarily processed and next digested in conditions simulating the human digestive tract (one of the digestive tracts was equipped with a mechanism simulating absorption). Soy tempeh stimulated most the growth of bacteria of the genus Bifidobacterium, while bean tempeh that of Escherichia coli. Using simulation of absorption for the digestion of fried soy tempeh resulted in a higher rise in the bacteria count of the genus Lactobacillus, while after digestion of fried bean tempeh the highest increase was recorded for Bifidobacterium and E. coli.

Antagonistic activity of Bacillus subtilis strains isolated from various sources

2018 ◽  
Vol 8 (2) ◽  
pp. 354-364
Author(s):  
A. N. Irkitova ◽  
A. V. Grebenshchikova ◽  
A. V. Matsyura
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Wild Strain ◽  
Fish Farming ◽  
Antagonistic Activity ◽  
Antagonistic Effect ◽  
E Coli ◽  
Long Period ◽  
Test Culture ◽  
Agar Media

<p>An important link in solving the problem of healthy food is the intensification of the livestock, poultry and fish farming, which is possible only in the adoption and rigorous implementation of the concept of rational feeding of animals. In the implementation of this concept required is the application of probiotic preparations. Currently, there is an increased interest in spore probiotics. In many ways, this can be explained by the fact that they use no vegetative forms of the bacilli and their spores. This property provides spore probiotics a number of advantages: they are not whimsical, easily could be selected, cultivated, and dried. Moreover, they are resistant to various factors and could remain viable during a long period. One of the most famous spore microorganisms, which are widely used in agriculture, is <em>Bacillus subtilis</em>. Among the requirements imposed to probiotic microorganisms is mandatory – antagonistic activity to pathogenic and conditional-pathogenic microflora. The article presents the results of the analysis of antagonistic activity of collection strains of <em>B. subtilis</em>, and strains isolated from commercial preparations. We studied the antagonistic activity on agar and liquid nutrient medias to trigger different antagonism mechanisms of <em>B. subtilis</em>. On agar media, we applied three diffusion methods: perpendicular bands, agar blocks, agar wells. We also applied the method of co-incubating the test culture (<em>Escherichia coli</em>) and the antagonist (or its supernatant) in the nutrient broth. Our results demonstrated that all our explored strains of <em>B. subtilis</em> have antimicrobial activity against a wild strain of <em>E. coli</em>, but to varying degrees. We identified strains of <em>B. subtilis</em> with the highest antagonistic effect that can be recommended for inclusion in microbial preparations for agriculture.</p><p><em><br /></em><em></em></p>

Synthesis of Chalcones and Nucleosides Incorporating [1, 3, 4]Oxadiazolenone Core and Evaluation of their Antifungal and Antibacterial Activities

2020 ◽  
Vol 15 (6) ◽  
pp. 665-679
Author(s):  
Alok K. Srivastava ◽  
Lokesh K. Pandey
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Aspergillus Niger ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Good Antibacterial Activity

Background: [1, 3, 4]oxadiazolenone core containing chalcones and nucleosides were synthesized by Claisen-Schmidt condensation of a variety of benzaldehyde derivatives, obtained from oxidation of substituted 5-(3/6 substituted-4-Methylphenyl)-1, 3, 4-oxadiazole-2(3H)-one and various substituted acetophenone. The resultant chalcones were coupled with penta-O-acetylglucopyranose followed by deacetylation to get [1, 3, 4] oxadiazolenone core containing chalcones and nucleosides. Various analytical techniques viz IR, NMR, LC-MS and elemental analysis were used to confirm the structure of the synthesised compounds.The compounds were targeted against Bacillus subtilis, Staphylococcus aureus and Escherichia coli for antibacterial activity and Aspergillus flavus, Aspergillus niger and Fusarium oxysporum for antifungal activity. Methods: A mixture of Acid hydrazides (3.0 mmol) and N, Nʹ- carbonyl diimidazole (3.3 mmol) in 15 mL of dioxane was refluxed to afford substituted [1, 3, 4]-oxadiazole-2(3H)-one. The resulted [1, 3, 4]- oxadiazole-2(3H)-one (1.42 mmol) was oxidized with Chromyl chloride (1.5 mL) in 20 mL of carbon tetra chloride and condensed with acetophenones (1.42 mmol) to get chalcones 4. The equimolar ratio of obtained chalcones 4 and β -D-1,2,3,4,6- penta-O-acetylglucopyranose in presence of iodine was refluxed to get nucleosides 5. The [1, 3, 4] oxadiazolenone core containing chalcones 4 and nucleosides 5 were tested to determined minimum inhibitory concentration (MIC) value with the experimental procedure of Benson using disc-diffusion method. All compounds were tested at concentration of 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.62 mg/mL, 0.31 mg/mL and 0.15 mg/mL for antifungal activity against three strains of pathogenic fungi Aspergillus flavus (A. flavus), Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum) and for antibacterial activity against Gram-negative bacterium: Escherichia coli (E. coli), and two Gram-positive bacteria: Staphylococcus aureus (S. aureus) and Bacillus subtilis(B. subtilis). Result: The chalcones 4 and nucleosides 5 were screened for antibacterial activity against E. coli, S. aureus and B. subtilis whereas antifungal activity against A. flavus, A. niger and F. oxysporum. Compounds 4a-t showed good antibacterial activity whereas compounds 5a-t containing glucose moiety showed better activity against fungi. The glucose moiety of compounds 5 helps to enter into the cell wall of fungi and control the cell growth. Conclusion: Chalcones 4 and nucleosides 5 incorporating [1, 3, 4] oxadiazolenone core were synthesized and characterized by various spectral techniques and elemental analysis. These compounds were evaluated for their antifungal activity against three fungi; viz. A. flavus, A. niger and F. oxysporum. In addition to this, synthesized compounds were evaluated for their antibacterial activity against gram negative bacteria E. Coli and gram positive bacteria S. aureus, B. subtilis. Compounds 4a-t showed good antibacterial activity whereas 5a-t showed better activity against fungi.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

Toxicity of the First Ten MEIC Chemicals to Bacteria

1993 ◽  
Vol 21 (2) ◽  
pp. 151-155
Author(s):  
Gustaw Kerszman
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Linear Regression ◽  
Minimal Inhibitory Concentration ◽  
Blood Concentration ◽  
Inhibitory Concentration ◽  
Human Lymphocytes ◽  
Bacterial Species ◽  
E Coli ◽  
Mild Toxicity

The toxicity of the first ten MEIC chemicals to Escherichia coli and Bacillus subtilis was examined. Nine of the chemicals were toxic to the bacteria, with the minimal inhibitory concentration (MIC) ranging from 10-3 to 4.4M. The sensitivities of both organisms were similar, but the effect on E. coli was often bactericidal, while it was bacteriostatic for B. subtilis. Digoxin was not detectably toxic to either bacterial species. Amitriptyline and FeSO4 were relatively less toxic to the bacteria than to human cells. For seven chemicals, a highly significant linear regression was established between log MIC in bacteria and log of blood concentration, giving lethal and moderate/mild toxicity in humans, as well as with toxicity to human lymphocytes.

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

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