Activity Of Gram-negative Bacteria Research Articles

Discovery of vinylogous carbamates as a novel class of β-ketoacyl-acyl carrier protein synthase III (FabH) inhibitors

β-Ketoacyl-acyl carrier protein synthase III (FabH) catalyzes the initial step of fatty acid biosynthesis via a type II fatty acid synthase in most bacteria. The important role of this essential enzyme combined with its unique structural features and ubiquitous occurrence in bacteria has made it an attractive new target for the development of new FabH inhibitors. We first used a structure-based approach to develop 24 new vinylogous carbamates ( 4a– 15a, 4b– 15b) that target FabH for the development of new antibiotics in this paper. Potent FabH inhibitory and selective anti- Gram-negative bacteria activities were observed in most of these vinylogous carbamates. Especially, compound 6a and 7a showed the most potent FabH inhibitory activity with IC 50 of 2.6 and 3.3 μM, respectively. Docking simulation was performed to position compound 6a into the Escherichia coli FabH active site and the possible binding conformation of compounds has been proposed. The biological data and molecular docking indicated that compounds 6a and 7a were potent inhibitors of E. coli FabH as antibiotics deserving further research.

Earthworms indirectly reduce the effects of take-all ( Gaeumannomyces graminis var. tritici) on soft white spring wheat ( Triticum aestivum cv. Fielder)

Earthworms have been shown to decrease the severity of some soil borne plant diseases including take-all ( Gaeumannomyces graminis var. tritici ( Ggt)). However, the mechanism by which the disease is affected by earthworms remains unknown. We conducted both field and controlled-environment experiments to examine the interactions between earthworms and Ggt. The controlled-environment experiments used split-pots in all possible combinations of with or without earthworms and with or without Ggt on either side of a semi-permeable membrane. Phospholipid fatty acid (PLFA) analysis was used to study gross changes in the microbial community associated with these treatments. The results from the field experiments showed that earthworms increased the yield, but had no effect on the severity of Ggt in soft white spring wheat. Similarly, in the controlled-environment experiments Aporrectodea tuberculata increased plant growth, but had no effect on the severity of Ggt. There was a significant increase in the percent total N and a lower C:N ratio regardless of earthworms in Ggt infested soil compared with non-infested soil. The PLFA analysis showed that earthworms increased the total amount of PLFA in soil and the activity of Gram negative bacteria. This indicated that there was a larger and more active microbial population when earthworms were present. The PLFA ratio of bacteria: fungi showed a significant interaction between earthworms and take-all. Earthworms increased the fungal biomass in the Ggt inoculated treatment and in contrast, promoted the bacteria when there was no Ggt. The mechanism by which earthworms reduce the severity of take-all and increase the growth of soft white spring wheat appeared to be microbially-mediated.

Effect of lipid A on neonatal and adult red blood cell deformation

Lipid A is responsible for the endotoxic activities of gram-negative bacteria. Red blood cell (RBC) deformation was studied in adults, in full-term and preterrn infants during 60 min of in vitro incubation with lipid A (1 00 ~g/ml) using a rheoscope. Compared to control values without lipid A incubation, RBC deformation (shear stress of 4 Pa) in adults, preterm, and from full-term neonates were decreased after 15 min (11%, 10%, 8%, respectively), after 30 min (11%, 6%, 3%, respectively) and after 60 min (8%, 5%, 2%, respectively) of incubation with lipid A. The decrease in RBC deformation after 15 min was significant (p<0.05) in the four groups. After 30 and 60 min, RBC deformation in the neonates recovered to the control values. In adults, RBC deformation improved after 30 and 60 min but did not reach preincubation values. These results indicate that neonatal RBC deformability recovers more rapidly than adult RBC during lipid A incubation.

Lipid A Binding to Neonatal and Adult Red Blood Cells

Lipid A is responsible for the endotoxic activities of gram-negative bacteria. Binding of lipid A (50 micrograms/ml) to RBC was studied using a passive hemolysis test. RBC from adults, cord and venous RBC from full-term infants and RBC from preterm infants were studied. Lipid A sensitized RBC were hemolysed with anti-lipid A and guinea pig complement. Hemolysis was expressed as hemoglobin concentration (absorbance at 546 nm) in the supernatant after centrifugation. 50 micrograms/ml lipid A did not increase spontaneous hemolysis (< 3%) after 60 min of incubation in any of the four groups. The passive hemolysis test did not result in additional hemolysis (5%) of umbilical cord RBC. RBC of preterm infants showed a significant increase in hemolysis (24%) after 60 min of incubation. In RBC of full-term neonates, increased hemolysis (14 and 46%) was detected after 30 and 60 min of incubation. Adult RBC hemolysed stronger (26 and 64%) after 30 and 60 min than neonatal RBC. We conclude that lipid A binds less to neonatal RBC compared to adults.

Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria.

In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria. In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions. Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iterons at the replication origin. Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria. Substitution of a serine at that position preserved limited function in replication and DNA binding. The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function. Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin iterons but caused a complete loss of replication activity in all four bacteria. Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting iteron binding activity. However, substitution of this arginine with a lysine resulted in a loss of replication activity in E. coli and A. vinelandii but had no effect in P. putida and A. tumefaciens. These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its iteron binding activity.

Din+ ('invertase') activity in Gram-negative bacteria

Din+ ('invertase') activity in Gram-negative bacteria

Proteolytic activities of gram-negative bacteria isolated from human periodontal lesions.

Enzymatic activity of gram-negative bacteria isolated from human periodontal lesions were examined in this study. The cells of Bacteroides gingivalis showed caseinolytic, trypsin-like, collagenase and immunoglobulin-degradating activities. The caseinolytic activity of black-pigmented Bacteroides species was found both in the culture supernatant and membraneous component of cells. No strains tested possessed strong mucopolysaccharidase activity. Capnocytophaga strains showed lipase (C14) and trypsin-like activities. No other strains of Bacteroides, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum or Eikenella corrodens exhibited any proteolytic activity.

Phospholipase D activity of gram-negative bacteria.

A phospholipase hydrolyzing cardiolipin to phosphatidic acid and phosphatidyl glycerol was characterized in gram-negative bacteria but was absent in preparations of gram-positive bacteria, Saccharomyces cerevisiae, and rat liver mitochondria. In cell-free extracts of Escherichia coli, Salmonella typhimurium, Proteus vulgaris, and Pseudomonase aeruginosa, this cardiolipin-hydrolyzing enzyme had similar pH and Mg2+ requirements and displayed a specificity which excluded phosphatidyl glycerol and phosphatidyl ethanolamine as substrates.

Arylamidase of Neisseria catarrhalis.

Neisseria catarrhalis produces arylamidase intracellularly and is one of the gram-negative bacteria producing exceptionally large amounts of this enzyme. In general, gram-positive bacteria do not produce this enzyme. Arylamidase from N. catarrhalis was purified by salt fractionation, chromatography, and density gradient ultracentrifugation. Its sedimentation coefficient was 6.6; l-alanine-beta-naphthylamide (betaNA) was the most rapidly hydrolyzed amino acid-betaNA. The enzyme had pK(e) values of 6.1 and 8.7 and pK(es) values of 7.1 and 7.9; only those amino acid-betaNA compounds of the l configuration were susceptible to hydrolysis. Arylamidase catalyzed stepwise hydrolysis of dipeptide-betaNA, beginning with the N-terminal residue. Substrates having amino acid residues with larger R groups, such as leucine, interacted much more effectively with enzyme. The significance of the predominate occurrence of arylamidase activity in gram-negative bacteria and the role of this enzyme in the physiology of these organisms remain unclear. It has been established, however, that arylamidase is distinct from leucine aminopeptidase.