Production and use of monoclonal antibodies for identification of Bradyrhizobium japonicum strains

1988 ◽  
Vol 34 (1) ◽  
pp. 88-92 ◽  
Author(s):  
D. Velez ◽  
J. D. Macmillan ◽  
L. Miller
Keyword(s):  
Monoclonal Antibodies ◽  
Bradyrhizobium Japonicum ◽  
Chemical Nature ◽  
Periodate Oxidation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
Polyclonal Antisera ◽  
Somatic Antigens

Thirteen murine hybridomas capable of producing monoclonal antibodies to somatic antigens on Bradyrhizobium japonicum were developed and an indirect enzyme-linked immunosorbent assay was used to test reactivity of the antibodies against 20 strains of B. japonicum. Although polyclonal antisera from mice immunized with strains of B. japonicum reacted with bacterial cells of all 20 strains, individual monoclonals were more specific. Some antibodies reacted with as few as 2 and one with as many as 11 strains. On the basis of reactivity with the set of 13 monoclonal antibodies, the 20 strains of B. japonicum could be divided arbitrarily into five groups. Three of five monoclonal antibodies tested reacted with bacteroids taken directly from soybean nodules. One monoclonal bound to cells of five species of Rhizobium, but none of the 13 reacted with gram-negative bacteria representing six other genera. Treatment of cells with reagents and heat indicated the chemical nature of the antigens to five of the monoclonals. Antigen reactive with one antibody was destroyed by periodate oxidation indicating that it was a polysaccharide. Two antigens were probably proteins as they could be digested by trypsin and denatured by heat. Two others were inactivated by all three treatments suggesting they were glycoproteins.

Monoclonal antibodies that identify gram-negative bacteria using the magnetic immunoluminescence assay

1992 ◽  
Vol 15 (2) ◽  
pp. 135-142 ◽  
Author(s):  
Ruurd Torensma ◽  
Marit J.C. Visser ◽  
Colinda J.M. Aarsman ◽  
Anja Groebbé-Heij ◽  
Miriam J.J.G. Poppelier ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Gram Negative Bacteria ◽  
Gram Negative

scholarly journals Chimeric Analysis of the Multicomponent Multidrug Efflux Transporters from Gram-Negative Bacteria

2002 ◽  
Vol 184 (23) ◽  
pp. 6499-6507 ◽  
Author(s):  
Elena B. Tikhonova ◽  
Quiju Wang ◽  
Helen I. Zgurskaya
Keyword(s):  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Multidrug Efflux ◽  
Structural Determinants ◽  
Gram Negative ◽  
E Coli ◽  
Multidrug Transporters ◽  
Cytoplasmic Membranes ◽  
Hybrid Genes ◽  
Rnd Transporters

ABSTRACT Many multidrug transporters from gram-negative bacteria belong to the resistance-nodulation-cell division (RND) superfamily of transporters. RND-type multidrug transporters have an extremely broad substrate specificity and protect bacterial cells from the actions of antibiotics on both sides of the cytoplasmic membrane. They usually function as three-component assemblies spanning the outer and cytoplasmic membranes and the periplasmic space of gram-negative bacteria. The structural determinants of RND transporters responsible for multidrug recognition and complex assembly remain unknown. We constructed chimeric RND transporters composed of N-terminal residues of AcrB and C-terminal residues of MexB, the major RND-type transporters from Escherichia coli and Pseudomonas aeruginosa, respectively. The assembly of complexes and multidrug efflux activities of chimeric transporters were determined by coexpression of hybrid genes either with AcrA, the periplasmic component of the AcrAB transporter from E. coli, or with MexA and OprM, the accessory proteins of the MexAB-OprM pump from P. aeruginosa. We found that the specificity of interaction with the corresponding periplasmic component is encoded in the T60-V612 region of transporters. Our results also suggest that the large periplasmic loops of RND-type transporters are involved in multidrug recognition and efflux.

scholarly journals Specific and Rapid Enumeration of Viable but Nonculturable and Viable-Culturable Gram-Negative Bacteria by Using Flow Cytometry

2010 ◽  
Vol 76 (15) ◽  
pp. 5088-5096 ◽  
Author(s):  
Mohiuddin M. Taimur Khan ◽  
Barry H. Pyle ◽  
Anne K. Camper
Keyword(s):  
Flow Cytometry ◽  
Pseudomonas Syringae ◽  
Phase 1 ◽  
Epifluorescence Microscopy ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Culture Methods ◽  
Viable But Nonculturable ◽  
Gram Negative ◽  
Analytical Technique

ABSTRACT An issue of critical concern in microbiology is the ability to detect viable but nonculturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population, and other options (direct viable count and the double-staining method using epifluorescence microscopy and inhibitory substance-influenced molecular methods) are also biased and time-consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella enterica serovar Typhimurium cells after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40, and propidium iodide (PI). The FCM data were compared with those for specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were nonculturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell membranes and were therefore theoretically dead. Data obtained using four different Gram-negative bacteria exposed to heat and stained with PI also illustrate the usefulness of the approach for the rapid and unbiased detection of dead versus live organisms.

scholarly journals Plants Secrete Substances That Mimic Bacterial N-Acyl Homoserine Lactone Signal Activities and Affect Population Density-Dependent Behaviors in Associated Bacteria

2000 ◽  
Vol 13 (6) ◽  
pp. 637-648 ◽  
Author(s):  
Max Teplitski ◽  
Jayne B. Robinson ◽  
Wolfgang D. Bauer
Keyword(s):  
Chemical Nature ◽  
Higher Plants ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Gram Negative Bacteria ◽  
Associated Bacteria ◽  
Gram Negative ◽  
Density Dependent ◽  
Reporter Strains ◽  
And Behavior

In gram-negative bacteria, many important changes in gene expression and behavior are regulated in a popula tion density-dependent fashion by N-acyl homoserine lac tone (AHL) signal molecules. Exudates from pea (Pisum sativum) seedlings were found to contain several separable activities that mimicked AHL signals in well-characterized bacterial reporter strains, stimulating AHL-regulated be haviors in some strains while inhibiting such behaviors in others. The chemical nature of the active mimic com pounds is currently unknown, but all extracted differently into organic solvents than common bacterial AHLs. Various species of higher plants in addition to pea were found to secrete AHL mimic activities. The AHL signal-mimic compounds could prove to be important in determining the outcome of interactions between higher plants and a diver sity of pathogenic, symbiotic, and saprophytic bacteria.

Epitope mapping of monoclonal antibodies specific for the directly cross-linkedmeso-diaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacteriumPectinatus cerevisiiphilus

1999 ◽  
Vol 45 (9) ◽  
pp. 779-785 ◽  
Author(s):  
Barry Ziola ◽  
Sheryl L Gares ◽  
Brandene Lorrain ◽  
Lori Gee ◽  
W M Ingledew ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Outer Membrane ◽  
Epitope Mapping ◽  
Sodium Dodecyl ◽  
Diaminopimelic Acid ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Spoilage Bacteria ◽  
Beer Spoilage ◽  
Bacterial Outer Membrane

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.Key words: beer spoilage, epitope mapping, monoclonal antibodies, Pectinatus, peptidoglycan.

scholarly journals Bacterial Type VI secretion system could have evolved from co-opted tail sheath tube of bacteriophages

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Protein Structure ◽  
Bacterial Genome ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Type Vi Secretion System ◽  
Gram Negative ◽  
Type Vi Secretion ◽  
Phage Tail ◽  
Tail Protein

Bacterial cells utilize a variety of nanomachines to secrete proteins and other molecules into the extracellular environment or target cells. One example is the Type VI secretion system (T6SS) in Gram-negative bacteria. Armed with a contractile mechanism similar to that used by bacteriophages to inject phage DNA into bacterial cells, the T6SS shares a common evolutionary origin with tail associated proteins of bacteriophages at both the structural and protein composition levels. Specifically, proteins constituting the T6SS are known to share provenance with those of the phage tail protein. More importantly, the T6SS is strikingly similar to the phage tail protein in both structure and function. However, a more important question concerns whether the T6SS evolved from the phage tail protein and if yes, what is the mechanism responsible for its development? One possibility could be the co-opt of the tail protein structure by bacterial cells through integration of the genes encoding the tail protein structure within the bacterial genome. In this case, expression of the phage tail protein genes would have resulted in a multiprotein structure without apparent function, which meant that a significant gap remains in comparison with extant T6SS that spans the inner and outer cell membrane of Gram-negative bacteria. While it is desirable to trace the evolutionary steps taken by phage tail proteins to transform into functional T6SS, multiple selection pressure and strong mutational propensity might have erased molecular evidence of such transformation. Hence, the challenge lies in uncovering as much structural and sequence evidence as possible that points to distinct steps in the evolutionary pathway towards T6SS. Structural studies offer a particularly promising route to unentangle the details but it must be augmented with sequence evidence that pins down the molecular events that shape the evolution of the complex multiprotein structure, where clefts from one protein fit into the folds of another in yielding a function that could evolve over eons. Collectively, structural and functional similarity between T6SS and phage tail protein suggests a common evolutionary origin for both macromolecular complexes, which has been established through combined structural, compositional and sequence analysis. But the steps underpinning the transformation of phage tail protein into T6SS remain unclear, which obfuscate understanding of the evolutionary forces that shape the transformation. One possible evolutionary trajectory posits that genes expressing phage tail proteins were co-opted and integrated into the bacterial genome. However, significant gap remains between a phage tail protein structure with unclear function in the cytoplasm and a functional T6SS that spans two bacterial membranes. Future detective work at the structural and sequence level might offer clues to the evolutionary path trodden by a precursor of the bacterial T6SS.

Bacterial cell damage by the complement system of channel catfish, Ictalurus punctatus

1987 ◽  
Vol 45 ◽  
pp. 888-889
Author(s):  
Rosemarie Rosell-Davis ◽  
Jill A. Jenkins ◽  
Lewis B. Coons ◽  
Donald D. Ourth
Keyword(s):  
Ictalurus Punctatus ◽  
Complement System ◽  
Channel Catfish ◽  
Bacterial Cell ◽  
Cell Damage ◽  
Membrane Attack Complex ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
The Complement System

The alternative complement pathway (ACP) provides the non-immune channel catfish with protection against many Gram-negative bacteria. The role of serum complement against Gram-negative bacteria is death of cells by insertion of the membrane attack complex (C5b-9) into the cell membrane. The assembly of the membrane attack complex is generated by the ACP and is activated by bacterial cell wall components. Pseudomonas fluorescens is a pathogen of channel catfish. In this study, bacteria were examined after incubation with catfish serum by scanning and transmission electron microscopy (SEM and TEM) for ultrastructural evidence of cell envelope damage by the complement system.A percent bactericidal assay determined that catfish plasma was 99% bactericidal against a 24 h culture of P. fluorescens (ATCC 13525). Following a 1 h incubation at 30°C of bacterial dilutions with equal volumes of serum, heat-inactivated serum, zymosan-adsorbed serum, or saline, the bacterial cells were filtered onto 0.22 um nuclepore filters, fixed in glutaraldehyde, dehydrated in ethanol, critical point dried, sputter coated with 15 nm gold and imaged using a JEOL SEM.

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