Objectives: To investigate the role of the AdeABC multidrug efflux pump in the decreased susceptibility of clinical isolates of Acinetobacter calcoaceticus-Acinetobacter baumannii complex to tigecycline.Methods: Gene expression was analysed by Taqman RT-PCR. A single cross-over achieved insertional inactivation of the adeB gene with a suicide plasmid construct carrying an adeB fragment obtained by PCR. Analysis of the adeRS locus was performed by PCR and sequencing. Ribotyping was performed with the RiboPrinter system. MICs were determined by Etest.Results: Expression analysis revealed constitutive overexpression of adeABC in less-susceptible clinical isolates G5139 and G5140 (tigecycline MIC 5 4 mg/L) when compared with the isogenic clinical isolates G4904 and G5141 (MIC 5 1.5 mg/L). Insertional mutants GC7945 (adeB knockout in G5139) and GC7951 (adeB knockout in G5140) were obtained, which resulted in tigecycline MICs of 0.5 mg/L. As reported previously, the expression of adeABC is regulated by the two-component signalling system encoded by the adeR and adeS genes. PCR and sequencing analyses revealed an insertion of an IS ABA-1 element in the adeS gene of G5139 and G5140. Conclusions:The results of this study suggest that decreased susceptibility to tigecycline in the A. calcoaceticus-A. baumannii complex is associated with the overexpression of the AdeABC multidrug efflux pump.

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 g/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 IS903kan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 g/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.Tigecycline is an expanded broad-spectrum antibiotic representing a new class called the glycylcyclines. The glycylcyclines are semisynthetic derivatives of minocycline and have activity against many bacterial pathogens (2, 14, 15). It has been noted that a few species of gram-negative bacteria, including Pseudomonas aeruginosa, Proteus spp., Providencia spp., and Morganella morganii, are intrinsically less susceptible to tigecycline. Previous studies revealed the involvement of multidrug efflux systems such as MexXY and AcrAB in the decreased tigecycline susceptibility of P. aeruginosa and Proteus mirabilis, respectively (3, 22). These pumps belong to the resistance-nodulation-division (RND) family that combines bacterial transporters with a tripartite architecture and broad substrate specificity (9, 12). Due to the broad substrate specificity of RND pumps, their overexpression usually results in the multidrug resistance (MDR) phenotype.Klebsiella pneumoniae causes infections of wounds, the urinary tract, and the respiratory system. This bacterial species is generally susceptible to tigecycline; however, a few clinical strains with decreased tigecycline susceptibility have been isolated. In this study, one such an isolate, G340, was investigated to determine the mechanism of decreased tigecycline susceptibility in K. pneumoniae.( MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains and plasmids used in this study are shown in Table 1. Strains...

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 hepatitis C virus (HCV)1 has caused a global crisis by infecting more than 2% of the world's population (1). Combination therapies using interferon and the antiviral drug ribavirin eliminate HCV in a majority of patients but such treatments are costly and cause debilitating side effects. Vaccine and drug development has been difficult because the only natural hosts for HCV are humans and chimpanzees, and the virus cannot be conventionally cultivated in vitro. As a result, the enzymes encoded by the virus have been intensely studied for rational drug design (2). Several groups have reported potent compounds that inhibit the HCV RNA-dependent RNA polymerase (3) and its serine protease (4). However, few inhibitors of the HCV helicase have been explored as antivirals because less is known about the mechanism of action of the HCV helicase and if or how it differs from similar cellular enzymes.HCV helicase is part of the viral non-structural protein 3 (NS3). The N-terminal one-third of NS3 is a chymotrypsin-like serine protease, and the C-terminal NS3 portion is a helicase that is fueled by the hydrolysis of ATP. Although the HCV helicase only unwinds duplex RNA during viral replication, the purified protein unwinds DNA dramatically more efficiently than RNA (5). In these reactions, HCV helicase requires a 3Ј single-stranded (ss) DNA or RNA overhang to initiate unwinding and is therefore classified as a 3Ј-5Ј helicase. The structure of the HCV helicase has been determined four times using x-ray crystallography: twice as an apoenzyme (6, 7), once with a bound ssDNA (8) and once as part of the full-length NS3 protein (9). NMR structures of parts of the HCV helicase have also been reported (10, 11). HCV helicase consists of 3 domains, with ATP likely binding between the structurally similar domains 1 and 2. One strand of the unwound duplex binds in the cleft that separates domain 3 from domains 1 and 2 (8). Gorbalenya and Koonin (12) have classified the HCV helicase as a member of helicase superfamily 2, which contains several large protein families that all share several signature sequences. The SF2 helicase signature sequence motifs line the "ATP-binding" cleft between domains 1 and 2. Numerous studies have elucidated the roles of the SF2 motifs in HCV helicase function, and structure-based mutagenesis has revealed a few other critical conserved residues (reviewed in Refs. 2 and 13). Little is known about the function of residues outside these conserved motifs.In the present study, sequence alignments and structurebased site-directed mutagenesis were used to identify two additional conserved motifs in domain 2 of the HCV helicase that are critical for helicase action. After introducing individual point mutations into these regions of the enzyme, ATPase, unwinding, translocation r...

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

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.The hepatitis C virus (HCV) evolves so rapidly that, in individual patients infected with certain genotypes, HCV exists as a heterogeneous population of quasispecies. Both viral genotype and quasispecies diversity influence disease severity and treatment response (reviewed in references 9 and 45). Patients infected with genotype 1 often do not respond to antiviral therapy, whereas patients with other genotypes respond more favorably (25). The kinetics of viral load decreases after the initiation of interferon therapy suggested a high freevirion clearance rate, a low rate of virus production in infected cells, and a high rate of infected-cell death in patients infected with genotype 2 relative to those in patients infected with genotype 1 (28). Strains that more rapidly evolve into diverse quasispecies more readily evade the host immune system, leading to chronic hepatitis (10), and respond less well to therapeutic intervention (11). Exactly how HCV genetic variation leads to these clinically important viral phenotypes is still largely a mystery, however. This comparative study was therefore initiated to define the impact of genetic variation on the activity of one of the best characterized HCV proteins, the NS3 helicase.HCV is a positive-sense, single-stranded RNA (ssRNA), and as such, when it enters a cell, HCV genomic RNA is translated into a peptide over 3,000 amino acids long from a single open reading frame. Both the host and viral proteases process the HCV polyprotein into three structural proteins (core, E1, and E2) and seven nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Most studies dealing with the impact of genetic variation on the HCV life cycle have focused on linking heterogeneity in NS5A (7) and E2 (38) with the tre...

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing ␣-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.

This study suggested that a loss of MarR functionality due to a frameshift mutation resulted in constitutive overproduction of MarA and AcrAB and, consequently, in decreased susceptibility to tigecycline in clinical isolates of E. coli.

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 ⍀ 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 M 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 M, 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.Chlorinated hydrocarbons are used for a variety of uses, including degreasing of metal parts, scouring of textiles, and the production of organic chemicals and pharmaceuticals. Due to their extensive uses coupled with both accidental and purposeful releases, these compounds are commonly found in subsurface soils and groundwater (38,45). Although these compounds are refractory, they have been shown to be degraded through a variety of microbially mediated processes. Specifically, under anaerobic conditions, many of these compounds have been shown to be reductively dechlorinated (6,15,25,33,34,42). Under aerobic conditions, some of these compounds can be utilized as growth substrates (12, 24) or more commonly cooxidized in conjunction with a growth substrate such as methane, ammonia, toluene, or phenol (3, 4, 17-21, 30, 32, 36, 37, 41, 43, 44). Of the cells capable of growth on these substrates, methanotrophs are often used for the degradation of chlorinated hydrocarbons due to their ubiquity (23).In methanotrophs, the enzyme responsible for chlorinated solvent degradation, methane monooxygenase (MMO), is also responsible for the initial oxidation of the sole gro...