The antibacterial effects of tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. Characterization of the minimal inhibitory concentration (MIC) of oxacillin for 30 S. aureus strains isolated from patients treated with oxacillin identified 13 strains with an oxacillin MIC C 4 lg/mL as methicillinresistant Staphylococcus aureus (MRSA) (range: 8 to 512 lg/ mL), while 17 strains were methicillin-susceptible Staphylococcus aureus (MSSA) (range: 0.25-0.5 lg/mL). The MICs of TPP ranged from 50 to 180 lg/mL for both the MSSA and the MRSA strains. The MICs of oxacillin for each of the 13 MRSA strains were reduced between 8-and 128-fold when these strains were coincubated with sub-MIC (B0.59 MIC) levels of TPP, demonstrating that the combination of TPP plus oxacillin was synergistic for all of the clinical MRSA isolates. Two-dimensional polyacrylamide gel electrophoresis identified 14 extracellular proteins of MRSA-13 down-regulated and 3 proteins up-regulated by exposure to TPP. These studies demonstrate that TPP can differentially stimulate the expression of various proteins in these bacteria and synergize the bactericidal activity of oxacillin for MRSA.
The purpose of this work was to investigate the induction of stress shock proteins in Burkholderia sp. YK-2 in response to the phenoxyherbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The stress shock proteins, which contribute to the resistance of the cytotoxic effect of 2,4-D, were induced at different 2,4-D concentrations in exponentially growing cultures of Burkholderia sp. YK-2. This response involved the induction of a 43-kDa DnaK and 41-kDa GroEL proteins, characterized by SDS-PAGE and Western blot by use of the anti-DnaK and anti-GroEL monoclonal antibodies. The total stress shock proteins were analyzed by 2-D PAGE. Survival of Burkholderia sp. YK-2 with time in the presence of different concentrations of 2,4-D was monitored, and viable counts paralleled the induction of the stress shock proteins in this strain.
BACKGROUND: Environmental contamination by nitroaromatic compounds such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-s-triazine (RDX), atrazine, and/or simazine (TRAS) generated as waste from military and agricultural activities is a serious worldwide problem. Microbiological treatment of these compounds is an attractive method because many explosives and herbicides are biodegradable and the process can be made cost-effective. We explored the feasibility of using cultures of Pseudomonas putida HK-6 for simultaneous degradation of TRAS with the aim of microbial application in wastewater treatment in bench-scale bioreactors.
Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V (max) and K ( m ) values of the enzyme for TNT were 12.6 micromol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain.
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