75 research outputs found
Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome
BACKGROUND: Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND) efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance. RESULTS: Genome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain. By reverse transcription (RT)-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump. CONCLUSION: The 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B. cenocepacia is an obligatory step prior to the design of specific, potent bacterial inhibitors for the improved control of infectious diseases. Consequently, the topic deserves to be further investigated and future studies will involve systematic investigation on the function and expression of each of the RND efflux pump homologs
Biological and structural characterization of theMycobacterium smegmatis nitroreductase NfnB, and its rolein benzothiazinone resistance
Tuberculosis is still a leading cause of death in developing
countries, for which there is an urgent need
for new pharmacological agents. The synthesis of
the novel antimycobacterial drug class of benzothiazinones
(BTZs) and the identification of their
cellular target as DprE1 (Rv3790), a component of
the decaprenylphosphoryl-b-D-ribose 2'-epimerase
complex, have been reported recently. Here, we
describe the identification and characterization of a
novel resistance mechanism to BTZ in Mycobacterium
smegmatis. The overexpression of the nitroreductase
NfnB leads to the inactivation of the drug by
reduction of a critical nitro-group to an amino-group.
The direct involvement of NfnB in the inactivation of
the lead compound BTZ043 was demonstrated by
enzymology, microbiological assays and gene knockout
experiments. We also report the crystal structure
of NfnB in complex with the essential cofactor flavin
mononucleotide, and show that a common amino
acid stretch between NfnB and DprE1 is likely to be
essential for the interaction with BTZ. We performed
docking analysis of NfnB-BTZ in order to understand
their interaction and the mechanism of
nitroreduction. Although Mycobacterium tuberculosis
seems to lack nitroreductases able to inactivate
these drugs, our findings are valuable for the design
of new BTZ molecules, which may be more effective
in vivo
Amino acid biosynthesis and its regulation in cyanobacteria.
Most of our knowledge of amino acid biosynthesis is implied from elegant studies on the biochemistry and molecular biology of bacterial amino acid biosynthesis. So far all this work deals with heterotrophic organisms completely dependent on their nutritional environment. Some researchers have therefore focused their attention on the problem, what happens to the regulation of a biosynthetic pathway during evolution to autotrophic growth. In cyanobacteria, there are few examples of metabolic controls by repression and de-repression of enzyme biosynthesis of the kind that are well documented for other microorganisms. In contrast, several cases have been reported demonstrating enzymic control by allosteric alterations. Many of the enzymes suspected to be involved in amino acid metabolism in cyanobacteria have not been isolated or characterized. Consequently, little is known of the molecular biology of these enzymes. This review will give a survey of information pertinent to study of amino acid biosynthesis and its regulation in cyanobacteria
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