Bifidobacterial β-Galactosidase-Mediated Production of Galacto-Oligosaccharides: Structural and Preliminary Functional Assessments

In the current study the ability of four previously characterized bifidobacterial β-galactosidases (designated here as BgaA, BgaC, BgaD, and BgaE) to produce galacto-oligosaccharides (GOS) was optimized. Of these enzymes, BgaA and BgaE were found to be promising candidates for GOS production (and the corresponding GOS mixtures were called GOS-A and GOS-E, respectively) with a GOS concentration of 19.0 and 40.3% (of the initial lactose), respectively. GOS-A and GOS-E were partially purified and structurally characterized. NMR analysis revealed that the predominant (non-lactose) disaccharide was allo-lactose in both purified GOS preparations. The predominant trisaccharide in GOS-A and GOS-E was shown to be 3′-galactosyllactose, with lower levels of 6′-galactosyllactose and 4′-galactosyllactose. These three oligosaccharides have also been reported to occur in human milk. Purified GOS-A and GOS-E were shown to be able to support bifidobacterial growth similar to a commercially available GOS. In addition, GOS-E and the commercially available GOS were shown to be capable of reducing Escherichia coli adhesion to a C2BBe1 cell line. Both in vitro bifidogenic activity and reduced E. coli adhesion support the prebiotic potential of GOS-E and GOS-A.

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Preliminary Characterization of the Transcriptional Response of the Porcine Intestinal Cell Line IPEC-J2 to EnterotoxigenicEscherichia coli,Escherichia coli, andE. coliLipopolysaccharide

Comparative and Functional Genomics ◽

10.1155/2010/469583 ◽

2010 ◽

Vol 2010 ◽

pp. 1-11 ◽

Cited By ~ 24

Author(s):

Marisa M. Geens ◽

Theo A. Niewold

Keyword(s):

Escherichia Coli ◽

Cell Line ◽

Transcriptional Response ◽

Intestinal Cell ◽

Adhesion Assay ◽

Transcriptional Level ◽

Suitable Model ◽

E Coli ◽

Intestinal Cell Line

IPEC-J2, a promisingin vitromodel system, is not well characterized especially on the transcriptional level, in contrast to human counterparts. The aim of this study was to characterize the gene expression in IPEC-J2 cells when coincubated with enterotoxigenicEscherichia coli(ETEC), nonpathogenicE. coli, andE. coliendotoxin. Apical infection of polarized IPEC-J2 monolayers caused a time-dependent decrease in transepithelial electrical resistance (TEER). Microarray analysis showed up-regulation of interleukins when IPEC-J2 were cocultured withE. colistrains this has so far never been measured in this cell line. Highest IL8 expression was found with the ETEC strain possessing the F4 fimbrium, suggesting IPEC-J2 cells to be F4 receptor positive, confirmed in a brush border membrane adhesion assay. It is concluded that the innate immune responses to pathogens and LPS makes the IPEC-J2 cell line a suitable model for research on intestinal host pathogen interaction.

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Bacteriostasis of Escherichia coli by milk: I. Colonization of breast-fed infants by milk resistant organisms

Journal of Hygiene ◽

10.1017/s0022172400055960 ◽

1977 ◽

Vol 78 (1) ◽

pp. 85-93 ◽

Cited By ~ 12

Author(s):

Jean M. Dolby ◽

Pauline Honour ◽

H. B. Valman

Keyword(s):

Escherichia Coli ◽

Human Milk ◽

Bacteriostatic Effect ◽

Gram Negative ◽

E Coli ◽

Resistant Strains ◽

Gram Negative Organisms ◽

Breast Fed

SUMMARYHuman milk has a bacteriostatic effect on Escherichia coli in vitro. The milks of 40 mothers were tested for this effect against E. coli isolated from their stools, from those of their own babies, and from those of babies not breast-fed. The milks had a direct bacteriostatic effect, not dependent on complement, on some but not all the strains of E. coli. Breast-fed babies receiving supplementary bottle feeds were colonized with milk-resistant strains, whereas bottle-fed babies and, surprisingly, babies completely breast-fed were colonized equally with milk-sensitive and milk-resistant strains, as were the mothers. These results suggest that the bacteriostatic effect of human milk, demonstrable in vitro does sometimes operate in vivo.The antibacterial activity of human milk is not influenced by the O, H or K antigens of E. coli and is effective against other Gram-negative organisms, e.g. Salmonella, Klebsiella, Proteus.

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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

Current Organic Chemistry ◽

10.2174/1385272824999200812132256 ◽

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).

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In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

Scientific Reports ◽

10.1038/s41598-021-81140-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kaitlin S. Witherell ◽

Jason Price ◽

Ashok D. Bandaranayake ◽

James Olson ◽

Douglas R. Call

Keyword(s):

Escherichia Coli ◽

Antimicrobial Peptide ◽

Membrane Integrity ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Multidrug Resistant Bacteria ◽

E Coli ◽

Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

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Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

Biochemical Journal ◽

10.1042/bj2480043 ◽

1987 ◽

Vol 248 (1) ◽

pp. 43-51 ◽

Cited By ~ 17

Author(s):

J Charlier ◽

R Sanchez

Keyword(s):

Escherichia Coli ◽

Gene Product ◽

Activity Ratio ◽

Deae Cellulose ◽

Trna Synthetase ◽

Trna Synthetases ◽

E Coli ◽

Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

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A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽

10.1128/mbio.01105-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 7

Author(s):

Christopher W. Lennon ◽

Kimberly C. Lemmer ◽

Jessica L. Irons ◽

Max I. Sellman ◽

Timothy J. Donohue ◽

...

Keyword(s):

Escherichia Coli ◽

Structure Function ◽

Rhodobacter Sphaeroides ◽

Fatty Acid Content ◽

Regulatory Protein ◽

Growth Conditions ◽

Globular Domain ◽

Content Type ◽

E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

Biochemical Journal ◽

10.1042/bj20060038 ◽

2006 ◽

Vol 396 (3) ◽

pp. 565-571 ◽

Cited By ~ 28

Author(s):

Takaomi Nomura ◽

Kohji Nakano ◽

Yasushi Maki ◽

Takao Naganuma ◽

Takashi Nakashima ◽

...

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Synthetic Activity ◽

23S Rrna ◽

Elongation Factors ◽

Hybrid Form ◽

Pyrococcus Horikoshii ◽

E Coli ◽

Functional Features

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0·Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0·Ph-L12 complex and Ph-L11 could replace L10·L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

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Promiscuous Cross-seeding between Bacterial Amyloids Promotes Interspecies Biofilms

Journal of Biological Chemistry ◽

10.1074/jbc.m112.383737 ◽

2012 ◽

Vol 287 (42) ◽

pp. 35092-35103 ◽

Cited By ~ 61

Author(s):

Yizhou Zhou ◽

Daniel Smith ◽

Bryan J. Leong ◽

Kristoffer Brännström ◽

Fredrik Almqvist ◽

...

Keyword(s):

Escherichia Coli ◽

Shewanella Oneidensis ◽

Bacterial Attachment ◽

Surface Attachment ◽

E Coli ◽

Amyloid Aggregates ◽

Functional Amyloids ◽

Biofilm Development ◽

Distant Homolog

Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimers, Parkinsons, and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2, and Citrobacter koseri were able to cross-seed in vitro. The polymerization of Escherichia coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA was secreted from E. coli csgB− mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA was secreted by S. typhimurium csgB− mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.

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