Chromosomal Integration of Adenoviral Vector DNA In Vivo

So far there has been no report of any clinical or preclinical evidence for chromosomal vector integration following adenovirus (Ad) vector-mediated gene transfer in vivo. We used liver gene transfer with high-capacity Ad vectors in the FAH^Δexon5 mouse model to analyze homologous and heterologous recombination events between vector and chromosomal DNA. Intravenous injection of Ad vectors either expressing a fumarylacetoacetate hydrolase (FAH) cDNA or carrying part of the FAH genomic locus resulted in liver nodules of FAH-expressing hepatocytes, demonstrating chromosomal vector integration. Analysis of junctions between vector and chromosomal DNA following heterologous recombination indicated integration of the vector genome through its termini. Heterologous recombination occurred with a median frequency of 6.72 × 10⁻⁵ per transduced hepatocyte, while homologous recombination occurred more rarely with a median frequency of 3.88 × 10⁻⁷. This study has established quantitative and qualitative data on recombination of adenoviral vector DNA with genomic DNA in vivo, contributing to a risk-benefit assessment of the biosafety of Ad vector-mediated gene transfer.Recombinant adenovirus (Ad) vectors are under clinical development for different applications, including tumor therapy, vaccination, and gene therapy. Today, the largest number of clinical gene transfer trials has been based on Ad vectors (http://www.wiley.co.uk/genmed/clinical). Several Ad vectors are in phase III clinical trials, and two products have already been approved in China. The occurrence of malignancies due to retroviral integration and oncogene activation in a clinical trial for the treatment of children with SCID-X1 (10) has pointed to the need for a thorough preclinical evaluation of potential genotoxic effects due to chromosomal integration of gene transfer vectors as an important part of the overall risk-benefit analysis. Detailed information on genotoxicity following gene transfer is available for vectors derived from viruses of the Retroviridae and Parvoviridae families (2, 20, 23, 26, 46). Between 60 and 75% of integrations of retrovirus, lentivirus, or adeno-associated virus (AAV)-based vectors take place in or close to genes.Chromosomal integration of Ad vector DNA following gene transfer in cell culture has been analyzed in only a few studies, and even less is known about Ad vector integration in vivo. Since the life cycle of wild-type adenovirus is extrachromosomal, Ad vectors are perceived to be nonintegrating vectors. However, in earlier studies it was observed that injection of hamsters with wild-type adenovirus type 12 (Ad12) resulted in tumor formation due to chromosomal integration of virus DNA and expression of the E1A/E1B oncoproteins (33). Recent in vitro studies with Ad vectors with E1 deletions have demonstrated the occurrence of vector integration following transduction of transformed cell lines and primary cells, with the frequencies of homologous and heterologous recombination being between 10⁻³ and 10⁻⁶ and between 10⁻³ and 10⁻⁵ per cell, respectively, depending on the conditions used (12, 14, 28, 36, 37, 42, 43). Since clinical gene transfer trials, including prophylactic vaccination of healthy volunteers against infectious diseases, are performed with large amounts of vector (in general, between 10¹⁰ and 10¹³ particles), it is possible that substantial integration of adenoviral vector DNA might also occur in vivo even if integration rates were low. However, so far there has been no attempt to experimentally address the issue of Ad vector integration in vivo. We used the FAH^Δexon5 mouse model (8) of tyrosinemia type I (MIM 27670) to analyze potential homologous and heterologous recombination events between Ad vector DNA and chromosomal DNA in vivo. Tyrosinemia type I is caused by the lack of fumarylacetoacetate hydrolase, an enzyme that is involved in the tyrosine degradation pathway and that converts fumarylacetoacetate into fumaric acid and acetoacetic acid in hepatocytes (38). Loss of fumarylacetoacetate hydrolase (FAH) activity in hepatocytes results in the accumulation of toxic and mutagenic metabolites in a cell-autonomous fashion, leading after birth to an acute hepatopathy and later in life to a chronic hepatopathy. Liver damage can be prevented both in humans and in FAH-deficient animals by the administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks the tyrosine degradation pathway by inhibiting 4-hydroxphenyl pyruvate dioxygenase, thereby preventing the accumulation of the toxic compounds. The murine FAH gene is located on chromosome 7, contains 14 exons, and spans 20.5 kb.The autosomal recessive FAH^Δexon5 mouse model, in which exon 5 is disrupted by the insertion of a NeoR gene (8), has been a useful system to analyze chromosomal integration of AAV, retrovirus, Sleeping Beauty transposon, and plasmid DNA in hepatocytes (13, 25, 27, 31). Similar to human tyrosinemia type I patients with spontaneous reversions of point mutations (18), FAH-expressing hepatocytes have a strong growth advantage over FAH^−/− hepatocytes, and the developing nodules, consisting of FAH-positive [FAH⁺] hepatocytes, can be easily distinguished in an environment of FAH^−/− hepatocytes. Following injection of an FAH-expressing Ad vector with the E1 deletion (30) into FAH^−/− mice, the development of FAH⁺ nodules in the livers of the experimental animals was observed, suggesting potential chromosomal integration of vector DNA. Since transgene expression from vectors with the E1 deletion is transient, in part due to viral toxicity and an immune response directed to viral proteins expressed from the vector, integration events and their characterization were not possible. We reasoned that the use of high-capacity Ad (HC-Ad) vectors (also called “helper-dependent” or “gutless” Ad vectors) (41) not expressing any viral proteins would allow reliable data on Ad vector integration in vivo to be obtained.     

In Vitro Analysis of Virus Particle Subpopulations in Candidate Live-Attenuated Influenza Vaccines Distinguishes Effective from Ineffective Vaccines

Two effective (vac⁺) and two ineffective (vac⁻) candidate live-attenuated influenza vaccines (LAIVs) derived from naturally selected genetically stable variants of A/TK/OR/71-delNS1[1-124] (H7N3) that differed only in the length and kind of amino acid residues at the C terminus of the nonstructural NS1 protein were analyzed for their content of particle subpopulations. These subpopulations included total physical particles (measured as hemagglutinating particles [HAPs]) with their subsumed biologically active particles of infectious virus (plaque-forming particles [PFPs]) and different classes of noninfectious virus, namely, interferon-inducing particles (IFPs), noninfectious cell-killing particles (niCKPs), and defective interfering particles (DIPs). The vac⁺ variants were distinguished from the vac⁻ variants on the basis of their content of viral subpopulations by (i) the capacity to induce higher quantum yields of interferon (IFN), (ii) the generation of an unusual type of IFN-induction dose-response curve, (iii) the presence of IFPs that induce IFN more efficiently, (iv) reduced sensitivity to IFN action, and (v) elevated rates of PFP replication that resulted in larger plaques and higher PFP and HAP titers. These in vitro analyses provide a benchmark for the screening of candidate LAIVs and their potential as effective vaccines. Vaccine design may be improved by enhancement of attributes that are dominant in the effective (vac⁺) vaccines.Live-attenuated vaccines are considered more effective than their inactive or single-component counterparts because they activate both the innate and adaptive immune systems and elicit responses to a broader range of antigens for longer periods of time (2, 10, 25, 28). Influenza virus variants with alterations in the reading frame of the nonstructural NS1 protein gene (delNS1), which express truncated NS1 proteins, characteristically induce enhanced yields of type I interferon (IFN) relative to the yields of their isogenic parental virus encoding full-length NS1 proteins (11, 13, 21, 33, 39). Many of these delNS1 variants have proved to be effective as live-attenuated influenza vaccines (LAIVs), providing protection against challenge virus in a broad range of species (33, 46), including chickens (39, 44). The IFN-inducing capacity of the virus is considered an important element in the effectiveness of LAIVs (33). In that context, influenza viruses are intrinsically sensitive to the antiviral action of IFN (31, 32, 36), although they may display a nongenetic-based transient resistance (36). In addition, IFN sensitizes cells to the initiation of apoptosis by viruses (42) and by double-stranded RNA (40), which may be spontaneously released in the course of influenza virus replication (14). Furthermore, IFN functions as an adjuvant to boost the adaptive immune response in mammals (3, 4, 11, 26, 41, 43, 46) and in chickens when administered perorally in the drinking water of influenza virus-infected birds (19). This raises the question: does the enhanced induction of IFN by delNS1 variants suffice to render an infectious influenza virus preparation sufficiently attenuated to function as an effective live vaccine? To address that question, we turned to a recent report that described the selection of several variants of influenza virus with a common backbone of A/TK/OR/71-SEPRL (Southeast Poultry Research Laboratory) that contained NS1 protein genes which were unusual in the length and nature of the amino acid residues at the C termini of the truncated NS1 proteins that they expressed because of the natural introduction of a frameshift and stop codon by the deletion in the NS1 protein gene (44). delNS1 variants were isolated from serial low-inoculum passages of TK/OR/71-delNS1[1-124] (H7N3) in eggs (44). Four of these genetically stable plaque-purified variants, each encoding a truncated NS1 protein of a particular length, were tested as a candidate LAIV in 2-week-old chickens. Two of the delNS1 variants were effective as live vaccines (double deletions [D-del] pc3 and pc4) (phenotypically vac⁺), and two were not (D-del pc1 and pc2) (phenotypically vac⁻) (44), despite only subtle differences in their encoded delNS1 proteins. Why were they phenotypically different?The present study addresses this question by analyzing and comparing the different virus particles that constitute the subpopulations of these two effective (vac⁺) and two ineffective (vac⁻) live vaccine candidates. These analyses are based on recent reports in which noninfectious but biologically active particles (niBAPs) in subpopulations of influenza virus particles were defined and quantified (20, 21, 29). The study described in this report reveals several quantitative and qualitative differences between the particle subpopulations of the four candidate LAIVs, including the different types of IFN-induction dose-response curves, the quantum (maximum) yields (QY) of IFN induced, the efficacy of the interferon-inducing particles (IFPs), the replication efficiency of the virus, and the size of the plaques that they produced. Evidence is presented that the in vitro analysis of virus particle subpopulations may be useful to distinguish vac⁺ from vac⁻ LAIV candidates and provide a basis for identifying and enhancing the performance of particles with desirable phenotypes.     

Respiration of Escherichia coli Can Be Fully Uncoupled via the Nonelectrogenic Terminal Cytochrome bd-II Oxidase

The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.The aerobic respiratory chain of Escherichia coli can function with a variety of different membrane-bound NADH dehydrogenases, including NDH-I, NDH-II, and WrbA (8, 26-28), as well as YhdH and QOR (15, 38, 39), on the electron input side and three ubiquinol oxidases (cytochromes bd-I, bd-II, and bo) (12, 14, 19, 22, 29) on the output side (Fig. ​(Fig.1).1). The stoichiometry for the number of protons pumped for each two electrons transferred (H⁺/2e⁻ ratio) has unequivocally been determined for NDH-I (H⁺/2e⁻, 4) and NDH-II (H⁺/2e⁻, 0) (10, 23, 41). Although no specific data are available for WrbA, YhdH, and QOR, it is generally assumed that these NADH:quinone oxidoreductases are not electrogenic because of the absence of (predicted) transmembrane alpha-helices (15, 38, 39). Similarly, the energy-conserving efficiencies of the cytochrome bd-I oxidase and the cytochrome bo oxidase are different; the cytochrome bd-I complex does not actively pump protons, but due to the oxidation of the quinol on the periplasmic side of the membrane and subsequent uptake of protons from the cytoplasmic side of the membrane, which are used in the formation of water, net electron transfer results in proton translocation with an H⁺/2e⁻ stoichiometry of 2 (32). In contrast, the cytochrome bo complex actively pumps protons over the membrane, resulting in an H⁺/2e⁻ stoichiometry of 4 (33, 42). The stoichiometry of proton translocation of the cytochrome bd-II complex is unknown.Open in a separate windowFIG. 1.Diagram of all NADH:quinone oxidoreductases and quinol:oxygen oxidoreductases in E. coli and their proton translocation properties. Cyt, cytochrome; Q, quinone.Due to the differences in the H⁺/e⁻ ratios of the dehydrogenases involved, two-electron transfer from NADH to the quinone pool may be accompanied by the translocation of any number of protons between 0 and 4, and subsequent reoxidation of the quinol pool may contribute to proton translocation again with a stoichiometry that depends on the relative activities of the terminal oxidases. The loose coupling between energy conservation and electron flow in respiration has been interpreted as a physiological means for the cell to cope with sudden changes in the rate of electron influx into the respiratory chain and/or in the availability of terminal electron acceptors on its terminal side (10). The fact that this energetic efficiency can vary is of great interest, both for understanding the physiological adaptive responses of the microbial cell and for biotechnological applications (e.g., synthesis of any oxidized compound with minimal biomass production). For this, it is important to quantify the flux distribution over and the efficiencies of the components of the respiratory machinery in relation to environmental conditions.Previous studies (10) have shown that NDH-I, NDH-II, and the two well-characterized cytochrome oxidases contribute significantly to the overall electron flux and furthermore that the distribution of fluxes over these components depends on environmental conditions, such as the growth rate in glucose-limited chemostats (10). In addition, it has been suggested that the flux distribution over the terminal oxidases of E. coli is dependent on the culture pH (40). However, the cytochrome bd-II oxidase was not taken into account in these previous studies.Here we present data that show that cytochrome bd-II oxidase participates significantly in oxygen reduction both during nonlimited growth in batch cultures and in glucose-limited chemostat cultures. For further quantification of the contribution of the respiratory chain to oxidative phosphorylation, it is essential to assess the in vivo H⁺/2e⁻ stoichiometry of the cytochrome bd-II oxidase (4, 37). Essentially, the approach used in previous studies by Calhoun et al. (10) was followed: strains with respiratory chains that were modified such that their H⁺/2e⁻ stoichiometry was fixed and known were grown under identical, glucose-limited conditions in chemostat culture. A flux analysis with respect to glucose catabolism and respiration allowed calculation of the rate of ATP synthesis for these strains. The data were then used as reference flux data for a strain that contained the cytochrome bd-II oxidase as the sole terminal oxidase. This strain showed a decreased yield with respect to oxygen and glucose. In this way we demonstrated that electron flow through the cytochrome bd-II oxidase does not contribute to the generation of a proton motive force. The results are discussed in view of the biochemical characterization of the enzyme and its physiological importance to adaptive responses by E. coli to an ever-changing environment.     

Requirement of JIP1-Mediated c-Jun N-Terminal Kinase Activation for Obesity-Induced Insulin Resistance

The c-Jun NH₂-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr¹⁰³ replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.Diet-induced obesity causes insulin resistance and metabolic syndrome, which can lead to β-cell dysfunction and type 2 diabetes (15). It is established that feeding mice a high-fat diet (HFD) causes activation of c-Jun NH₂-terminal kinase 1 (JNK1) (10). Moreover, Jnk1^−/− mice are protected against the effects of HFD-induced insulin resistance (10). Together, these observations indicate that JNK1 plays a critical role in the metabolic stress response. However, the mechanism that accounts for HFD-induced JNK1 activation is unclear. Recent studies have implicated the JIP1 scaffold protein in JNK1 activation caused by metabolic stress (23, 39).JIP1 can assemble a functional JNK activation module composed of a mitogen-activated protein kinase (MAPK) kinase kinase (a member of the mixed-lineage protein kinase [MLK] group), the MAPK kinase MKK7, and JNK (40, 42). This complex may be relevant to JNK activation caused by metabolic stress (23, 39). Indeed, MLK-deficient mice (14) and JIP1-deficient mice (13) exhibit defects in HFD-induced JNK activation and insulin resistance.The protection of Jip1^−/− mice against the effects of being fed an HFD may be mediated by loss of the JNK scaffold function of JIP1. However, JIP1 has also been reported to mediate other biochemical processes that would also be disrupted in Jip1^−/− mice. For example, JIP1 interacts with AKT and has been implicated in the mechanism of AKT activation (8, 17, 18, 34). Moreover, JIP1 interacts with members of the Src and Abl tyrosine kinase families (4, 16, 24), the lipid phosphatase SHIP2 (44), the MAPK phosphatase MKP7 (43), β-amyloid precursor protein (20, 31), the small GTPase regulatory proteins Ras-GRF1, p190-RhoGEF, RalGDS, and Tiam1 (2, 8, 21), ankyrin G (35), molecular chaperones (35), and the low-density-lipoprotein-related receptors LRP1, LRP2, and LRP8 (7, 37). JIP1 also interacts with other scaffold proteins, including the insulin receptor substrate proteins IRS1 and IRS2 (35). Finally, JIP1 may act as an adapter protein for kinesin-mediated (11, 12, 16, 38, 42) and dynein-mediated (35) trafficking on microtubules. The JNK scaffold properties of JIP1 therefore represent only one of the possible biochemical functions of JIP1 that are disrupted in Jip1^−/− mice.The purpose of this study was to test the role of JIP1 as a JNK scaffold protein in the response of mice to being fed an HFD. Our approach was to examine the effect of a point mutation that selectively prevents JIP1-induced JNK activation. It is established that phosphorylation of JIP1 on Thr¹⁰³ is required for JIP1-mediated JNK activation by the MLK pathway (25). Consequently, the phosphorylation-defective Thr103Ala JIP1 protein does not activate JNK (25). Here we describe the analysis of mice with a point mutation in the Jip1 gene that replaces the JIP1 phosphorylation site Thr¹⁰³ with Ala. We show that this mutation suppresses HFD-induced JNK activation and insulin resistance. These data demonstrate that JNK activation mediated by the JIP1 scaffold complex contributes to the response of mice to an HFD.     

Microbial and Physicochemical Characteristics of Compact Anaerobic Ammonium-Oxidizing Granules in an Upflow Anaerobic Sludge Blanket Reactor

Anaerobic ammonium oxidation (anammox) is a promising new process to treat high-strength nitrogenous wastewater. Due to the low growth rate of anaerobic ammonium-oxidizing bacteria, efficient biomass retention is essential for reactor operation. Therefore, we studied the settling ability and community composition of the anaerobic ammonium-oxidizing granules, which were cultivated in an upflow anaerobic sludge blanket (UASB) reactor seeded with aerobic granules. With this seed, the start-up period was less than 160 days at a NH₄⁺-N removal efficiency of 94% and a loading rate of 0.064 kg N per kg volatile suspended solids per day. The formed granules were bright red and had a high settling velocity (41 to 79 m h⁻¹). Cells and extracellular polymeric substances were evenly distributed over the anaerobic ammonium-oxidizing granules. The high percentage of anaerobic ammonium-oxidizing bacteria in the granules could be visualized by fluorescent in situ hybridization and electron microscopy. The copy numbers of 16S rRNA genes of anaerobic ammonium-oxidizing bacteria in the granules were determined to be 4.6 × 10⁸ copies ml⁻¹. The results of this study could be used for a better design, shorter start-up time, and more stable operation of anammox systems for the treatment of nitrogen-rich wastewaters.The anaerobic ammonia oxidation (anammox) process is a recently discovered biological nitrogen removal technology in which ammonia is oxidized to nitrogen gas with nitrite as the electron acceptor (5, 29, 32). In contrast to heterotrophic denitrification (6, 26), the anammox process does not require external electron donors (e.g., methanol) due to their chemolithoautotrophic lifestyle. Furthermore, if this process is combined with a partial nitrification step, only half of the ammonium needs to be nitrified to nitrite, which together with the remaining ammonium can subsequently be converted into nitrogen through the anammox process. This reduces the oxygen demand of the system and leads to further reduction in operational costs (27).The anaerobic ammonium-oxidizing bacteria (anammox bacteria) have a low growth rate (18), with a doubling time at best estimated as 7 to 11 days (18, 28). The yield of the anammox bacteria has been determined to be 0.066 mol C biomass mol⁻¹ ammonium consumed, and the maximum ammonium consumption rate is ∼45 nmol mg⁻¹ protein min⁻¹ (18). Given the low growth rate and low yield, very efficient biomass retention is essential to retain the anammox bacteria within the reactor systems during cultivation (19). The enrichment of anammox bacteria from a mixed inoculum requires the optimization of conditions favorable for the anammox bacteria and generally takes 200 to 300 days (5, 6, 27). Thus, conditions that would reduce the start-up time of anammox reactors would positively effect the implementation of the process. Several sources of inocula, such as activated sludge (4), nitrifying activated sludge (27), and anaerobic sludge (6), have been used for the start-up of anammox reactors with start-up times of as long as 1,000 days (27).Aerobic granules have been reported to have high microbial diversity (31) and compact structure with very good settling properties resulting in an efficient means of biomass retention. These properties, including interspecies competition and mass transfer, result in the stratification of microbial species with anoxic pockets in the interior of the granules that may be suitable to harbor anammox bacteria. Therefore, the main objective of this study was to investigate the feasibility of start-up of the anammox process by seeding the reactor with aerobic granular sludge by using an upflow anaerobic sludge blanket (UASB) reactor. After the successful start-up and the formation of anammox granules, the structure and physicochemical properties of the anammox granules and the reactor performance were characterized. Microbial community analysis revealed that the dominant anammox species was related to a species of anammox bacteria present in anammox biofilms.     

Impact of Therapeutic Treatment with β-Lactam on Transfer of the blaCTX-M-9 Resistance Gene from Salmonella enterica Serovar Virchow to Escherichia coli in Gnotobiotic Rats

The conjugative transfer of the plasmid carrying the bla_CTX-M-9 gene from Salmonella enterica serovar Virchow isolated from a chicken farm to a recipient Escherichia coli strain was evaluated in vitro and in axenic rats inoculated with both strains, with or without selective pressure due to therapeutic doses of cefixime. The transfer of the bla_CTX-M-9 gene of S. enterica serovar Virchow to E. coli was confirmed in vitro, at a low frequency of 5.9 × 10⁻⁸ transconjugants/donors. This transfer rate was higher in gnotobiotic rats and reached ∼10⁻⁵ transconjugants/donors without selective pressure. This frequency was not affected by the addition of therapeutic doses of cefixime. Thus, estimates of in vitro transfer underestimated potential transfer in the digestive tract, and therapeutic doses of cefixime did not increase the selection for transconjugants.β-Lactams and extended-spectrum cephalosporins are widely used in human and veterinary medicine to treat severe infections in humans and animals caused by Enterobacteriaceae and other gram-negative pathogens (7). However, extended-spectrum β-lactamases (ESBLs) have emerged and become the major mechanism of resistance to β-lactam antibiotics. Until the late 1990s, TEM and SHV enzymes were the predominant ESBLs. However, over the last decade, CTX-M-type enzymes have become the most prevalent extended-spectrum β-lactamases worldwide. Currently, there have been a number of reports documenting an increasing prevalence of enteric pathogens that produce these plasmid-mediated CTX-M enzymes (4, 8, 9, 39).Although originally confined to hospitals, ESBL-producing strains are now emerging in the community. Several investigations have shown that the rate of fecal carriage of ESBL-positive isolates (especially Escherichia coli) in humans is increasing, with CTX-M type enzymes found in most isolates (14, 17, 25, 28, 30, 36). This alarming phenomenon may have serious economic consequences and implications for treatment.bla_CTX-M genes spread throughout the community, mostly through the transmission of plasmids, and some studies have reported that animals may serve as a possible source for the dissemination of ESBL-encoding genes to humans. Indeed, common conjugative resistance plasmids and resistant clones have been found in animals, food products, and humans, suggesting that the transfer of extended-spectrum cephalosporin resistance between animals and humans is possible (17, 18, 21, 31, 40).In France, the emergence of the CTX-M-9 enzyme in Salmonella enterica serovar Virchow strains recovered from poultry, poultry products, and one human patient was reported between 2002 and 2003 (5, 31, 38, 40, 41). A comparative analysis of the bla_CTX-M-2 and bla_CTX-M-9 plasmids from S. enterica serovar Virchow isolates from human and poultry sources demonstrated a close relationship between the plasmids (16).To examine the hypothesis that antimicrobial resistance in humans could partly be attributed to food products, a number of studies have investigated the transfer of antibiotic resistance from bacteria originating in animal hosts or food products. Previously, Lester et al. (24) demonstrated the transfer of the vanA resistance gene from Enterococcus faecium isolated from animals to E. faecium isolated from human volunteers during transient intestinal colonization in humans in the absence of selective pressure. In a gnotobiotic mouse model, Feld et al. demonstrated the evidence of spread of a small plasmid pLFE1 harboring the erythromycin-resistant gene erm(B) from Lactobacillus plantarum isolated from raw-milk cheese to exogenous Enterococcus faecalis inoculated into the intestinal flora of mice (16). The transfer rate was enhanced when erythromycin was coadministered. Improvements in our understanding of the in vivo transmissibility and stability of antimicrobial resistance markers and the mechanisms driving horizontal transmission may help to predict the outcome of different strategies for controlling epidemic plasmids.The aim of the present study was to determine whether the bla_CTX-M-9 resistance gene could be transferred from an animal S. enterica serovar Virchow strain to a commensal E. coli strain that originated from the human intestinal tract while under selective pressure from a therapeutic dose of β-lactam. The transfer was investigated by using in vitro mating and a germfree rat model. We also evaluated the impact of the concentration of cefixime, an expanded-spectrum cephalosporin, on the transfer rate.     

Temporal Variation of Bacterial Respiration and Growth Efficiency in Tropical Coastal Waters

We investigated the temporal variation of bacterial production, respiration, and growth efficiency in the tropical coastal waters of Peninsular Malaysia. We selected five stations including two estuaries and three coastal water stations. The temperature was relatively stable (averaging around 29.5°C), whereas salinity was more variable in the estuaries. We also measured dissolved organic carbon and nitrogen (DOC and DON, respectively) concentrations. DOC generally ranged from 100 to 900 μM, whereas DON ranged from 0 to 32 μM. Bacterial respiration ranged from 0.5 to 3.2 μM O₂ h⁻¹, whereas bacterial production ranged from 0.05 to 0.51 μM C h⁻¹. Bacterial growth efficiency was calculated as bacterial production/(bacterial production + respiration), and ranged from 0.02 to 0.40. Multiple correlation analyses revealed that bacterial production was dependent upon primary production (r² = 0.169, df = 31, and P < 0.02) whereas bacterial respiration was dependent upon both substrate quality (i.e., DOC/DON ratio) (r² = 0.137, df = 32, and P = 0.03) and temperature (r² = 0.113, df = 36, and P = 0.04). Substrate quality was the most important factor (r² = 0.119, df = 33, and P = 0.04) for the regulation of bacterial growth efficiency. Using bacterial growth efficiency values, the average bacterial carbon demand calculated was from 5.30 to 11.28 μM C h⁻¹. When the bacterial carbon demand was compared with primary productivity, we found that net heterotrophy was established at only two stations. The ratio of bacterial carbon demand to net primary production correlated significantly with bacterial growth efficiency (r² = 0.341, df = 35, and P < 0.001). From nonlinear regression analysis, we found that net heterotrophy was established when bacterial growth efficiency was <0.08. Our study showed the extent of net heterotrophy in these waters and illustrated the importance of heterotrophic microbial processes in coastal aquatic food webs.As our understanding of the marine food web evolves, we recognize the importance of microorganisms in aquatic ecosystems. Bacteria are the main respirers and recycle a large pool of dissolved organic matter to higher trophic levels (6, 13). Therefore, bacterial production is a key process in dissolved organic matter flux. However, the transfer of dissolved organic matter to bacteria is more accurately reflected by bacterial carbon demand (BCD) or carbon consumption (23). One way to obtain BCD from bacterial production is through bacterial growth efficiency (BGE) or growth yield. BGE is an important parameter to evaluate the fate of organic carbon inputs and to determine whether bacteria act as a link (recyclers) or sink (mineralizers). Therefore, understanding the patterns of variation in BGE is fundamental for our knowledge of carbon cycling (14).BGE is essentially the ratio of carbon converted to biomass relative to all the carbon consumed, where carbon consumption is either measured as the sum of bacterial production and respiration (5, 24), dissolved organic matter utilization (3), or both (12). Although bacterial production is frequently measured, bacterial respiration measurements are still scarce (23) and are often derived from production rates assuming constant growth efficiency (7, 30). The use of a constant growth efficiency is, however, not valid in some situations as studies have shown that BGE varies over both time and space (8, 27, 28, 32).From cross-system compilations (15, 38, 49) and a comprehensive study in a temperate salt marsh estuary (4, 5), we begin to understand the factors that affect bacterial growth efficiency. Although substrate quantity and quality affect growth efficiency (4, 15), temperature is also an important factor (49). However, the effect of temperature differs for both bacterial production and bacterial respiration (5) and is distorted by substrate limitation (38).Most of the above studies are from temperate regions where there is marked seasonality in temperature. In temperate regions, the effects of temperature are usually more apparent (5, 32) and can sometimes distort the effects of other factors (4). Although temperature plays a major role in controlling heterotrophic activity in temperate regions, it plays a lesser role in the tropics, where temperatures are more stable and relatively higher. Tropical oceans cover about 40% of the global ocean (37), and yet knowledge of the structure and function of this ecosystem remains limited, especially in the region of Southeast Asia (29). Only a few related studies are available, and those are from tropical coastal waters in Goa, India (45), and mangrove and estuarine waters in Peninsular Malaysia (27, 28). Substrate quality is often suggested as a more important factor than temperature (27, 28, 45).In this paper, we addressed the following question: What is the effect of substrate quality and temperature toward BGE in tropical coastal waters? We measured bacterial respiration, production, and growth efficiency in tropical coastal waters and related their variation to changes in temperature and dissolved organic nutrient concentrations. Here, we show that although both temperature and substrate quality affected respiration, BGE was related to substrate quality only.     

Genome Sequences of Pelagibaca bermudensis HTCC2601T and Maritimibacter alkaliphilus HTCC2654T,the Type Strains of Two Marine Roseobacter Genera

Pelagibaca bermudensis HTCC2601^T and Maritimibacter alkaliphilus HTCC2654^T represent two marine genera in the globally significant Roseobacter clade of the Alphaproteobacteria. Here, we present the genome sequences of these organisms, isolated from the Sargasso Sea using dilution-to-extinction culturing, which offer insight into the genetic basis for the metabolic and ecological diversity of this important group.Organisms from the Roseobacter clade of the Alphaproteobacteria are numerically significant in the world''s oceans and have been found in a wide range of habitats (1, 3). Using previously described high-throughput dilution-to-extinction culturing (6, 13), the marine Roseobacter strains Pelagibaca bermudensis HTCC2601^T and Maritimibacter alkaliphilus HTCC2654^T were isolated in low-nutrient heterotrophic medium (LNHM) (4) from surface water collected at the Bermuda Atlantic Time-Series Study (BATS) site in the western Sargasso Sea (5, 9). As the type strains for two genera of this globally prolific Roseobacter group, P. bermudensis and M. alkaliphilus were selected for shotgun genome sequencing at the J. Craig Venter Institute through the Moore Foundation Microbial Genome Sequencing Project (http://www.moore.org/microgenome). Draft genomes of P. bermudensis and M. alkaliphilus, with 103 and 46 contigs, respectively, were annotated and analyzed through the Joint Genome Institute IMG/M website (http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) (10).The draft genomes of P. bermudensis and M. alkaliphilus comprise 5,425,920 and 4,529,231 bases, 5,522 and 4,764 predicted open reading frames (ORFs), and 66.44% and 64.13% G+C content, respectively. The P. bermudensis genome is predicted to contain 56 tRNA genes, five 5S rRNA genes, four 16S rRNA genes, and five 23S rRNA genes, and that of M. alkaliphilus 49 tRNA genes and one each of the 5S, 16S, and 23S rRNA genes. Both genomes have putative genes for complete glycolysis and Entner-Doudoroff pathways, a complete tricarboxylic acid cycle, and predicted metabolic pathways for the oxidation of C₁ compounds. Both have predicted genes for the synthesis of most essential amino acids and some vitamins and cofactors. Each has putative genes for the utilization of fructose, sucrose, and mannose, confirmed in physiological testing of P. bermudensis (5) but not for M. alkaliphilus (9). P. bermudensis contains a predicted complete RuBisCO complex, unique to the sequenced Roseobacter species (12, 15), a complete assimilatory nitrate reduction pathway, and several type VI secretion genes. M. alkaliphilus is predicted to have complete nitrate reduction pathways to both N₂ and ammonia and most type IV secretion genes. Both are predicted to have complete sec pathways and large numbers of ABC transporters (362 in P. bermudensis and 224 in M. alkaliphilus), similar to other Roseobacter strains (15).M. alkaliphilus was named because of its alkaline growth optimum at pH 10. Na⁺/H⁺ antiporters have been shown to be involved in conferring alkaliphilic phenotypes for a variety of organisms by increasing internal cellular H⁺ concentrations in alkaline conditions where Na⁺ is present (2, 7, 8, 14, 16, 17). As expected, the genome of M. alkaliphilus contains two putative Na⁺/H⁺ antiporters, one homologous to nhaP, important for alkaliphily in several strains (2, 16, 17), and another located adjacent to predicted ABC transporter genes for capsular polysaccharide export.     

Effect of Iron Concentration on the Growth Rate of Pseudomonas syringae and the Expression of Virulence Factors in hrp-Inducing Minimal Medium

Although chemically defined media have been developed and widely used to study the expression of virulence factors in the model plant pathogen Pseudomonas syringae, it has been difficult to link specific medium components to the induction response. Using a chemostat system, we found that iron is the limiting nutrient for growth in the standard hrp-inducing minimal medium and plays an important role in inducing several virulence-related genes in Pseudomonas syringae pv. tomato DC3000. With various concentrations of iron oxalate, growth was found to follow Monod-type kinetics for low to moderate iron concentrations. Observable toxicity due to iron began at 400 μM Fe³⁺. The kinetics of virulence factor gene induction can be expressed mathematically in terms of supplemented-iron concentration. We conclude that studies of induction of virulence-related genes in P. syringae should control iron levels carefully to reduce variations in the availability of this essential nutrient.The type III secretion system (T3SS) is used by diverse plant and animal pathogens to invade and colonize their hosts (1). This secretion system translocates bacterial proteins (effectors) from the bacterial cytoplasm directly into the eukaryotic host cell cytosol, where the effectors subvert host cell processes to the advantage of the pathogen. In Pseudomonas syringae pv. tomato DC3000, the T3SS is responsible for the elicitation of hypersensitive reactions of nonhost plants and is essential for disease on host plants (14). Many T3SS genes in plant pathogens are denoted hrp, for hypersensitive response and pathogenicity. We know of several regulatory elements that control T3SS genes in P. syringae pv. tomato DC3000 (7, 27), including HrpL, an alternative sigma factor. However, the exact environmental signals that the bacteria respond to are unknown.The expression of avrB, a T3SS effector, varies depending on the carbon source in Pseudomonas syringae pv. glycinea race 0 (9). Other environmental factors affecting the expression of virulence-related genes have also been studied. Nitrogen and osmolarity are important for the expression of the Pseudomonas syringae pv. syringae 61 hrp genes (28). Osmotic strength, pH, and carbon source differentially affected the expression of T3SS genes in Pseudomonas syringae pv. phaseolicola (18). These results imply that catabolite repression by the tricarboxylic acid cycle intermediates may be involved in the induction process. With other pathogenic bacteria, nutritional conditions are reported to be an important factor for the induction of virulence. For example, the Xanthomonas hrp genes are induced by sucrose and sulfur-containing amino acids (21). The optimal condition for hrp gene expression may simulate leaf apoplast environmental factors, including hypo-osmotic pressure, low pH, and limited nutrient concentration (18).Iron is a micronutrient (required in concentrations less than 10⁻⁴ M) for in vitro cultures (22), and the typical concentration needed for optimal bacterial growth is 0.3 to 1.8 μM (24). Iron is an essential element for bacteria due to its participation in the tricarboxylic acid cycle, electron transport, amino acid and pyrimidine biosynthesis, DNA synthesis, and other critical functions (3). Iron uptake must also be regulated due to its lethal effect through the Fenton reaction (2). The effect of iron limitation on bacterial growth has been documented for Escherichia coli cultures (6, 19, 20). Two studies have shown that production of the phytotoxins, syringomycin, and syringotoxin from P. syringae responds in batch culture to iron supplementation (5, 15). Iron is known to alter the physiology of other pseudomonads in both batch and chemostat cultures (11, 16). Although iron is the fourth most abundant element in the earth''s crust, its availability is very low due to its low solubility in aqueous solution ([Fe³⁺] at pH 7, 10⁻¹⁸ μM) (24). Bacteria have evolved complex mechanisms to ensure that iron requirements are met but not exceeded. Siderophore-mediated transport of iron is one of the mechanisms used by bacteria to uptake iron from their environment (17).In this study, medium components in hrp-inducing minimal medium were evaluated systematically with a chemostat culture. Iron was found to be both a growth-limiting nutrient in hrp-inducing minimal medium and a mediator of virulence gene expression in the model plant pathogen P. syringae pv. tomato DC3000.