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1.  Two Novel Conserved Motifs in the Hepatitis C Virus NS3 Protein Critical for Helicase Action* 
The Journal of biological chemistry  2003;278(45):44514-44524.
The hepatitis C virus (HCV) NS3 helicase shares several conserved motifs with other superfamily 2 (SF2) helicases. Besides these sequences, several additional helicase motifs are conserved among the various HCV genotypes and quasispecies. The roles of two such motifs are examined here. The first motif (YRGXDV) forms a loop that connects SF2 helicase motifs 4 and 5, at the tip of which is Arg393. When Arg393 is changed to Ala, the resulting protein (R393A) retains a nucleic acid stimulated ATPase but cannot unwind RNA. R393A also unwinds DNA more slowly than wild type and translocates poorly on single-stranded DNA (ssDNA). DNA and RNA stimulate ATP hydrolysis catalyzed by R393A like the wild type, but the mutant protein binds ssDNA more weakly both in the presence and absence of the non-hydrolyzable ATP analog ADP(BeF3). The second motif (DFSLDPTF) forms a loop that connects two anti-parallel sheets between SF2 motifs 5 and 6. When Phe444 in this Phe-loop is changed to Ala, the resulting protein (F444A) is devoid of all activities. When Phe438 is changed to Ala, the protein (F438A) retains nucleic acid-stimulated ATPase, but does not unwind RNA. F438A unwinds DNA and translocates on ssDNA at about half the rate of the wild type. Equilibrium binding data reveal that this uncoupling of ATP hydrolysis and unwinding is due to the fact that the F438A mutant does not release ssDNA upon ATP binding like the wild type. A model is presented explaining the roles of the Arg-clamp and the Phe-loop in the unwinding reaction.
PMCID: PMC3571693  PMID: 12944414
2.  Characterization and Sequence Analysis of Extended-Spectrum-β-Lactamase-Encoding Genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis Isolates Collected during Tigecycline Phase 3 Clinical Trials▿  
In concert with the development of novel β-lactams and broad-spectrum cephalosporins, bacterially encoded β-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-β-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the blaTEM, blaSHV, blaOXA, and blaCTX genes from clinical strains. Isolates were also screened for AmpC genes of the blaCMY, blaACT, blaFOX, and blaDHA families as well as the blaKPC genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of ≥2 μg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, blaCTX-M-type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas blaSHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC90 = 2 μg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.
PMCID: PMC2630642  PMID: 19015360
3.  Mixed Pollutant Degradation by Methylosinus trichosporium OB3b Expressing either Soluble or Particulate Methane Monooxygenase: Can the Tortoise Beat the Hare?▿  
Applied and Environmental Microbiology  2006;72(12):7503-7509.
Methanotrophs have been widely investigated for in situ bioremediation due to their ubiquity and their ability to degrade halogenated hydrocarbons through the activity of methane monooxygenase (MMO). It has been speculated that cells expressing the soluble form of MMO (sMMO) are more efficient in cleaning up sites polluted with halogenated hydrocarbons due to its broader substrate range and relatively fast degradation rates compared cells expressing the other form of MMO, the particulate MMO (pMMO). To examine this issue, the biodegradation of mixtures of chlorinated solvents, i.e., trichloroethylene (TCE), trans-dichloroethylene (t-DCE), and vinyl chloride (VC), by Methylosinus trichosporium OB3b in the presence of methane using either form of MMO was investigated over longer time frames than those commonly used, i.e., days instead of hours. Growth of M. trichosporium OB3b along with pollutant degradation were monitored and analyzed using a simple comparative model developed from the Ω model created for analysis of the competitive binding of oxygen and carbon dioxide by ribulose bisphosphate carboxylase. From these findings, it appears that at concentrations of VC, t-DCE, and TCE greater than 10 μM each, methanotrophs expressing pMMO have a competitive advantage over cells expressing sMMO due to higher growth rates. Despite such an apparent growth advantage, pMMO-expressing cells degraded less of these substrates at these concentrations than sMMO-expressing cells during active growth. If the concentrations were increased to 100 μM, however, not only did pMMO-expressing cells grow faster, they degraded more of these pollutants and did so in a shorter amount of time. These findings suggest that the relative rates of growth substrate and pollutant degradation are important factors in determining which form of MMO should be considered for pollutant degradation.
PMCID: PMC1694253  PMID: 17012599
4.  3,5-Dioxopyrazolidines, Novel Inhibitors of UDP-N- Acetylenolpyruvylglucosamine Reductase (MurB) with Activity against Gram-Positive Bacteria 
A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 μM, 4.3 to 10.3 μM, and 6.8 to 29.4 μM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 μM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 Å resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 μM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 μg/ml) and 4 (MICs, 4 to 8 μg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.
PMCID: PMC1366903  PMID: 16436710
5.  Influence of Transcriptional Activator RamA on Expression of Multidrug Efflux Pump AcrAB and Tigecycline Susceptibility in Klebsiella pneumoniae 
Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 μg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903φkan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 μg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 × 10−8. These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.
PMCID: PMC549240  PMID: 15728897
6.  AcrAB Efflux Pump Plays a Role in Decreased Susceptibility to Tigecycline in Morganella morganii 
Transposon mutagenesis of a clinical isolate of Morganella morganii, G1492 (tigecycline MIC of 4 μg/ml), yielded two insertion knockout mutants for which tigecycline MICs were 0.03 μg/ml. Transposon insertions mapped to acrA, which is constitutively overexpressed in G1492, suggesting a role of the AcrAB efflux pump in decreased susceptibility to tigecycline in M. morganii.
PMCID: PMC547285  PMID: 15673770
7.  Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli 
BMC Genomics  2003;4:36.
Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library.
Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport.
Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.
PMCID: PMC212392  PMID: 12964949
8.  Hepatitis C Virus NS3 ATPases/Helicases from Different Genotypes Exhibit Variations in Enzymatic Properties 
Journal of Virology  2003;77(7):3950-3961.
The NS3 ATPase/helicase was isolated and characterized from three different infectious clones of hepatitis C virus (HCV). One helicase was from a genotype that normally responds to therapy (Hel-2a), and the other two were from more resistant genotypes, 1a (Hel-1a) and 1b (Hel-1b). Although the differences among these helicases are generally minor, all three enzymes have distinct properties. Hel-1a is less selective for nucleoside triphosphates, Hel-1b hydrolyzes nucleoside triphosphates less rapidly, and Hel-2a unwinds DNA more rapidly and binds DNA more tightly than the other two enzymes. Unlike related proteins, different nucleic acid sequences stimulate ATP hydrolysis by HCV helicase at different maximum rates and with different apparent efficiencies. This nucleic acid stimulation profile is conserved among the enzymes, but it does not result entirely from differential DNA-binding affinities. Although the amino acid sequences of the three proteins differ by up to 15%, one variant amino acid that is critical for helicase action was identified. NS3 residue 450 is a threonine in Hel-1a and Hel-1b and is an isoleucine in Hel-2a. A mutant Hel-1a with an isoleucine substituted for threonine 450 unwinds DNA more rapidly and binds DNA more tightly than the parent protein.
PMCID: PMC150621  PMID: 12634355

Results 1-8 (8)