scholarly journals The primary step of biotin synthesis in mycobacteria

2020 ◽  
Vol 117 (38) ◽  
pp. 23794-23801
Author(s):  
Zhe Hu ◽  
John E. Cronan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Deletion Mutant ◽  
Essential Role ◽  
In Vivo Studies ◽  
Pimelic Acid ◽  
Chronic Infections ◽  
Acid Moiety ◽  
E Coli

Biotin plays an essential role in growth of mycobacteria. Synthesis of the cofactor is essential forMycobacterium tuberculosisto establish and maintain chronic infections in a murine model of tuberculosis. Although the late steps of mycobacterial biotin synthesis, assembly of the heterocyclic rings, are thought to follow the canonical pathway, the mechanism of synthesis of the pimelic acid moiety that contributes most of the biotin carbon atoms is unknown. We report that theMycobacterium smegmatisgene annotated as encoding Tam, anO-methyltransferase that monomethylates and detoxifiestrans-aconitate, instead encodes a protein having the activity of BioC, anO-methyltransferase that methylates the free carboxyl of malonyl-ACP. TheM. smegmatisTam functionally replacedEscherichia coliBioC both in vivo and in vitro. Moreover, deletion of theM. smegmatis tamgene resulted in biotin auxotrophy, and addition of biotin toM. smegmatiscultures repressedtamgene transcription. Although its pathogenicity precluded in vivo studies, theM. tuberculosisTam also replacedE. coliBioC both in vivo and in vitro and complemented biotin-independent growth of theM. smegmatis tamdeletion mutant strain. Based on these data, we propose that the highly conserved mycobacterial tamgenes be renamedbioC.M. tuberculosisBioC presents a target for antituberculosis drugs which thus far have been directed at late reactions in the pathway with some success.

scholarly journals Effects of Bacterial CLPB Protein Fragments on Food Intake and PYY Secretion

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2223
Author(s):  
Manon Dominique ◽  
Nicolas Lucas ◽  
Romain Legrand ◽  
Illona-Marie Bouleté ◽  
Christine Bôle-Feysot ◽  
...  
Keyword(s):  
Food Intake ◽  
Peptide Yy ◽  
Molecular Mimicry ◽  
Potential Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
E Coli ◽  
In Vivo Effects

CLPB (Caseinolytic peptidase B) protein is a conformational mimetic of α-MSH, an anorectic hormone. Previous in vivo studies have already shown the potential effect of CLPB protein on food intake and on the production of peptide YY (PYY) by injection of E. coli wild type (WT) or E. coli ΔClpB. However, until now, no study has shown its direct effect on food intake. Furthermore, this protein can fragment naturally. Therefore, the aim of this study was (i) to evaluate the in vitro effects of CLPB fragments on PYY production; and (ii) to test the in vivo effects of a CLPB fragment sharing molecular mimicry with α-MSH (CLPB25) compared to natural fragments of the CLPB protein (CLPB96). To do that, a primary culture of intestinal mucosal cells from male Sprague–Dawley rats was incubated with proteins extracted from E. coli WT and ΔCLPB after fragmentation with trypsin or after a heat treatment of the CLPB protein. PYY secretion was measured by ELISA. CLPB fragments were analyzed by Western Blot using anti-α-MSH antibodies. In vivo effects of the CLPB protein on food intake were evaluated by intraperitoneal injections in male C57Bl/6 and ob/ob mice using the BioDAQ® system. The natural CLPB96 fragmentation increased PYY production in vitro and significantly decreased cumulative food intake from 2 h in C57Bl/6 and ob/ob mice on the contrary to CLPB25. Therefore, the anorexigenic effect of CLPB is likely the consequence of enhanced PYY secretion.

scholarly journals Once-daily gentamicin therapy for experimental Escherichia coli meningitis.

1997 ◽  
Vol 41 (1) ◽  
pp. 49-53 ◽  
Author(s):  
A Ahmed ◽  
M M París ◽  
M Trujillo ◽  
S M Hickey ◽  
L Wubbel ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Bacterial Killing ◽  
Once Daily ◽  
E Coli ◽  
Aminoglycoside Therapy ◽  
Gentamicin Concentration ◽  
Gentamicin Therapy ◽  
The Impact

In vitro and in vivo studies have demonstrated that the bacteriologic efficacy of once-daily aminoglycoside therapy is equivalent to that achieved with conventional multiple daily dosing. The impact of once-daily dosing for meningitis has not been studied. Using the well-characterized rabbit meningitis model, we compared two regimens of the same daily dosage of gentamicin given either once or in three divided doses for 24 or 72 h. The initial 1 h mean cerebrospinal fluid (CSF) gentamicin concentration for animals receiving a single dose (2.9 +/- 1.7 micrograms/ml) was threefold higher than that for the animals receiving multiple doses. The rate of bacterial killing in the first 8 h of treatment was significantly greater for the animals with higher concentrations in their CSF (-0.21 +/- 0.19 versus -0.03 +/- 0.22 log10 CFU/ml/h), suggesting concentration-dependent killing. By 24h, the mean reduction in bacterial titers was similar for the two regimens. In animals treated for 72 h, no differences in bactericidal activity was noted for 24, 48, or 72 h. Gentamicin at two different dosages was administered intracisternally to a separate set of animals to achieve considerably higher CSF gentamicin concentrations. In these animals, the rate of bacterial clearance in the first 8 h (0.52 +/- 0.15 and 0.58 +/- 0.15 log10 CFU/ml/h for the lower and higher dosages, respectively) was significantly greater than that in animals treated intravenously. In conclusion, there is evidence of concentration-dependent killing with gentamicin early in treatment for experimental E. coli meningitis, and once-daily dosing therapy appears to be at least as effective as multiple-dose therapy in reducing bacterial counts in CSF.

scholarly journals The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

2003 ◽  
Vol 185 (10) ◽  
pp. 3076-3080 ◽  
Author(s):  
Dominic Esposito ◽  
Gary F. Gerard
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Essential Role ◽  
Bacteriophage Λ ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
In Vivo Recombination

ABSTRACT The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

scholarly journals MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and β-lactamase activities

Microbiology ◽  
2020 ◽  
Vol 166 (6) ◽  
pp. 546-553 ◽  
Author(s):  
Satya Deo Pandey ◽  
Diamond Jain ◽  
Neeraj Kumar ◽  
Anwesha Adhikary ◽  
Ganesh Kumar N. ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Type Species ◽  
Complex Structure ◽  
Ectopic Expression ◽  
Dual Function ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli

Mycobacterial peptidoglycan (PG) is an unsolved puzzle due to its complex structure and involvement of multiple enzymes in the process of its remodelling. dd-Carboxypeptidases are low molecular mass penicillin-binding proteins (LMM-PBPs) that catalyzes the cleavage of terminal d-Ala of muramyl pentapeptide branches and thereby helps in the PG remodelling process. Here, we have assigned the function of a putative LMM-PBP, MSMEG_2432 of Mycobacterium smegmatis , by showing that it exhibits both dd-CPase and β-lactamase activities. Like conventional dd-CPase (PBP5 from E. coli), upon ectopic complementation in a deformed seven PBP deletion mutant of E. coli, MSMEG_2432 has manifested its ability to restore ~75 % of the cell population to their normal rod shape. Further, in vitro dd-CPase assay has confirmed its ability to release terminal d-Ala from the synthetic tripeptide and the peptidoglycan mimetic pentapeptide substrates ending with d-Ala-d-Ala. Also, elevated resistance against penicillins and cephalosporins upon ectopic expression of MSMEG_2432 suggests the presence of β-lactamase activity, which is further confirmed in vitro through nitrocefin hydrolysis assay. Moreover, it is found apparent that D169A substitution in MSMEG_2432 influences both of its in vivo and in vitro dd-CPase and β-lactamase activities. Thus, we infer that MSMEG_2432 is a dual function enzyme that possesses both dd-CPase and β-lactamase activities.

scholarly journals In vitro antibiotic sensitivity and therapeutic efficacy of experimental salmonellosis, colibacillosis and pasteurellosis in broiler chickens

1970 ◽  
Vol 2 (2) ◽  
pp. 99-102 ◽  
Author(s):  
MA Rahman ◽  
MA Samad ◽  
MB Rahman ◽  
SML Kabir
Keyword(s):  
Broiler Chickens ◽  
Pasteurella Multocida ◽  
Antibiotic Sensitivity ◽  
Morbidity And Mortality ◽  
In Vivo Studies ◽  
Penicillin G ◽  
E Coli ◽  
Therapeutic Trials

Avian salmonellosis (AS), avian colibacillosis (AC) and avian pasteurellosis (AP) have been recognized as important bacterial diseases in poultry associated with morbidity and mortality in Bangladesh. The causative agents of these three diseases were isolated (5 isolates / disease) from dead chickens submitted for diagnosis at the BRAC Poultry Disease Diagnostic Centre, Gazipur during the period from January to December 2002. Five isolates of each of the Salmonella pullorum, Escherichia coli and Pasteurella multocida were evaluated against eight antibiotic containing disc which included ciprofloxacin, gentamicin, ampicillin, chloramphenicol, erythromycin, tetracycline, cephradine and penicillin G. Erythromycin in S. pullorum and Ciprofloxacin both in the E. coli and P. multocida were found highest sensitive, gentamicin, chloramphenicol, cephradine were found moderately sensitive to S. pullorum, gentamicin, tetracycline, erythromycin and ampicillin were found moderately sensitive to E. coli, and gentamicin ampicillin, cephradine and penicillin G were moderately sensitive to P. multocida. Therapeutic trials against experimentally produced S. pullorum, E. coli and P. multocida infection in three groups of broiler chickens showed that cephradine against S. pullorum and ciprofloxacin against both in E. coli and P. multocida were found highly effective both in vitro and in vivo studies, therefore, cephradine against salmonellosis and ciprofloxacin against colibacillosis and pasteurellosis are effective drugs of choice which could be used to control morbidity and mortality in poultry caused by these diseases.Key words: antibiotic sensitivity; salmonellosis; colibacillosis; pasteurellosis, broiler chickensdoi: 10.3329/bjvm.v2i2.2538Bangl. J. Vet. Med. (2004). 2 (2): 99-102

scholarly journals Dra/AfaE Adhesin of Uropathogenic Dr/Afa+Escherichia coli Mediates Mortality in Pregnant Rats

2005 ◽  
Vol 73 (11) ◽  
pp. 7597-7601 ◽  
Author(s):  
K. Wroblewska-Seniuk ◽  
R. Selvarangan ◽  
A. Hart ◽  
R. Pladzyk ◽  
P. Goluszko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Maternal Mortality ◽  
Double Mutant ◽  
Lethal Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
Pregnant Rats ◽  
E Coli

ABSTRACT Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE + afaD and afaE afaD + mutants. The clinical E. coli strain (afaE + afaD +) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE + afaD + strain, followed by the group infected with the afaE + afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE + strains were the most invasive (afaE + afaD strain > afaE + afaD + strain), while the afaE mutant strains (afaE afaD + and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE + E. coli strains in vitro.

scholarly journals Influence of the Sodium Salt of 3α,7α-Dihydroxy-12-Oxo-5β-Cholanate on Antimicrobial Activity of Ampicillin In Vitro

2018 ◽  
Vol 44 (1) ◽  
pp. 6
Author(s):  
Ljiljana Suvajdžić ◽  
Slobodan Gigov ◽  
Aleksandar Rašković ◽  
Srđan Stojanović ◽  
Maja Bekut ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Sodium Salt ◽  
Antimicrobial Effect ◽  
In Vivo Studies ◽  
Bacterial Suspension ◽  
Commercial Preparation ◽  
E Coli ◽  
Colony Forming Units

Background: Multiple resistances to antibiotics are an emergent problem worldwide. Scientists intensively search for new substances with the antimicrobial potential or the mode to restore the activity of old-generation antibiotics. Ampicillin is the antibiotic with the expanded range of antimicrobial activity, but its use has decreased due to the poor absorption and highly developed resistance. In vivo studies showed that ampicillin has better absorption and bioavailability if combined with bile acid salts. The aim of this study was to examine antimicrobial effects of ampicillin alone and its combination with semisynthetic monoketocholic acid salt (MKH) in vitro.Materials, Methods & Results: In this study, commercial preparation of ampicillin and sodium salt of 3α,7α-dihydroxy-12oxo-5β-cholanate were used. Their effects were evaluated on Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium), obtained from urine specimens of dogs with clinically manifested cystitis. The first two investigated strains were ampicillin-sensitive, while E. faecium was resistant to ampicillin. Modified macrodilution method according to Clinical and Laboratory Standards Institute Guidelines (M7-A8) was performed. Bacterial suspension equivalent to 0.5 McFarland was prepared in saline, compared to the standard (Biomerieux) ad oculi. The density was checked spectrophotometrically at a wavelength of 625 nm and adjusted if necessary to the desired absorbance from 0.08 to 0.1. The resultant suspension was diluted 1:100 and inoculated in test tubes. Number of bacteria was counted on Petri plates using dilutions from 10-3 to 10-7 in order to obtain valid and countable plates. One hundred microliters of appropriate dilutions were aseptically plated in triplicate onto nutrient agar. Plates were incubated on 37°C for 72 h, under aerobic conditions. The number of colony forming units (CFU) was determined by direct counting. As a valid for enumeration, we took plates with 30 to 300 CFU. Percentage of killed bacteria for ampicillin was from 69.33-95.19% for E. coli, 87.1296.92% for E. faecalis and 7.20-33.30% for E. faecium. Ampicillin applied in the combination with MKH killed 99.99% to 100% of E. coli, 94.59% to 99.91% of E. faecalis and 31.73% to 64.76% of E. faecium. Mean percentage of killed bacteria for ampicillin was 81.93% for E. coli, 91.64% for E. faecalis, and 18.13% for E. faecium, while in combination with MKH percentage was 99.96% for E. coli, 98.23% for E. faecalis and 47.54% for E. faecium.Discussion: Results are presented as pharmacological minimal inhibitory concentration (MIC) values. Ampicillin was applied at the concentration higher than the therapeutic one, which could explain high MIC values for E. coli and E. faecalis. The combination of ampicillin with MKH showed the best improvement of antimicrobial effect on E. faecium (Δ = 29.41%), isolate that was resistant to ampicillin when applied alone. In all the investigated isolates, the combinations with MKH were more effective than ampicillin administered alone. It seems that MKH demonstrates a synergistic antimicrobial activity with ampicillin in vitro, which considerably decreases MIC values for all investigated isolates. These results implicate that MKH could restore the previous activity of ampicillin against some bacteria, which could be a significant benefit for clinical practice.

scholarly journals The relA/spoT-Homologous Gene inStreptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

1998 ◽  
Vol 180 (16) ◽  
pp. 4123-4132 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Amino Acids ◽  
Deletion Mutant ◽  
Streptomyces Coelicolor ◽  
Stringent Response ◽  
Morphological Differentiation ◽  
Antibiotic Biosynthesis ◽  
Homologous Gene ◽  
E Coli

ABSTRACT Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined.S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence thatS. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. colitruncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relA in an S. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

scholarly journals Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis

2007 ◽  
Vol 404 (1) ◽  
pp. 115-120 ◽  
Author(s):  
Xue-Ming Xu ◽  
Bradley A. Carlson ◽  
Robert Irons ◽  
Heiko Mix ◽  
Nianxin Zhong ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Essential Role ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Nih3t3 Cells ◽  
Selenophosphate Synthetase ◽  
Interfering Rna

Selenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism.

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