Antimicrobial Activity of Simulated Solar Disinfection against Bacterial,Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m⁻²) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log₁₀ after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log₁₀ after 6 h) was obtained only in the presence of riboflavin and 250 W m⁻² irradiance.Solar disinfection (SODIS) is an established and proven technique for the generation of safer drinking water (11). Water is collected into transparent plastic polyethylene terephthalate (PET) bottles and placed in direct sunlight for 6 to 8 h prior to consumption (14). The application of SODIS has been shown to be a simple and cost-effective method for reducing the incidence of gastrointestinal infection in communities where potable water is not available (2-4). Under laboratory conditions using simulated sunlight, SODIS has been shown to inactivate pathogenic bacteria, fungi, viruses, and protozoa (6, 12, 15). Although SODIS is not fully understood, it is believed to achieve microbial killing through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating (21).The combination of UVA radiation and riboflavin (vitamin B₂) has recently been reported to have therapeutic application in the treatment of bacterial and fungal ocular pathogens (13, 17) and has also been proposed as a method for decontaminating donor blood products prior to transfusion (1). In the present study, we report that the addition of riboflavin significantly enhances the disinfectant efficacy of simulated SODIS against bacterial, fungal, and protozoan pathogens.Chemicals and media were obtained from Sigma (Dorset, United Kingdom), Oxoid (Basingstoke, United Kingdom), and BD (Oxford, United Kingdom). Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Candida albicans (ATCC 10231), and Fusarium solani (ATCC 36031) were obtained from ATCC (through LGC Standards, United Kingdom). Escherichia coli (JM101) was obtained in house, and the Legionella pneumophila strain used was a recent environmental isolate.B. subtilis spores were produced from culture on a previously published defined sporulation medium (19). L. pneumophila was grown on buffered charcoal-yeast extract agar (5). All other bacteria were cultured on tryptone soy agar, and C. albicans was cultured on Sabouraud dextrose agar as described previously (9). Fusarium solani was cultured on potato dextrose agar, and conidia were prepared as reported previously (7). Acanthamoeba polyphaga (Ros) was isolated from an unpublished keratitis case at Moorfields Eye Hospital, London, United Kingdom, in 1991. Trophozoites were maintained and cysts prepared as described previously (8, 18).Assays were conducted in transparent 12-well tissue culture microtiter plates with UV-transparent lids (Helena Biosciences, United Kingdom). Test organisms (1 × 10⁶/ml) were suspended in 3 ml of one-quarter-strength Ringer''s solution or natural freshwater (as pretreated water from a reservoir in United Kingdom) with or without riboflavin (250 μM). The plates were exposed to simulated sunlight at an optical output irradiance of 150 watts per square meter (W m⁻²) delivered from an HPR125 W quartz mercury arc lamp (Philips, Guildford, United Kingdom). Optical irradiances were measured using a calibrated broadband optical power meter (Melles Griot, Netherlands). Test plates were maintained at 30°C by partial submersion in a water bath.At timed intervals for bacteria and fungi, the aliquots were plated out by using a WASP spiral plater and colonies subsequently counted by using a ProtoCOL automated colony counter (Don Whitley, West Yorkshire, United Kingdom). Acanthamoeba trophozoite and cyst viabilities were determined as described previously (6). Statistical analysis was performed using a one-way analysis of variance (ANOVA) of data from triplicate experiments via the InStat statistical software package (GraphPad, La Jolla, CA).The efficacies of simulated sunlight at an optical output irradiance of 150 W m⁻² alone (SODIS) and in the presence of 250 μM riboflavin (SODIS-R) against the test organisms are shown in Table ​Table1.1. With the exception of B. subtilis spores and A. polyphaga cysts, SODIS-R resulted in a significant increase in microbial killing compared to SODIS alone (P < 0.001). In most instances, SODIS-R achieved total inactivation by 2 h, compared to 6 h for SODIS alone (Table ​(Table1).1). For F. solani, C. albicans, ands A. polyphaga trophozoites, only SODIS-R achieved a complete organism kill after 4 to 6 h (P < 0.001). All control experiments in which the experiments were protected from the light source showed no reduction in organism viability over the time course (results not shown).

TABLE 1.

Efficacies of simulated SODIS for 6 h alone and with 250 μM riboflavin (SODIS-R)

Bar-Coded Pyrosequencing of 16S rRNA Gene Amplicons Reveals Changes in Ileal Porcine Bacterial Communities Due to High Dietary Zinc Intake

Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.Zinc oxide (ZnO) is used as a feed additive for diarrhea prophylaxis in piglets (23). However, the mode of action of ZnO is not fully understood. Besides its effects on the host (10, 30, 31), high dietary zinc levels may affect the diversity of intestinal microbial communities (2, 11, 20). The prevention of postweaning diarrhea in piglets due to high dietary ZnO intake may not be directly related to a reduction of pathogenic E. coli (8) but, rather, to the diversity of the coliform community (15). Studies on the impact of high ZnO levels on the porcine ileal bacterial community are scarce but nevertheless important, as bacterial diarrhea is initiated in the small intestine (9, 17). The small intestine is a very complex habitat with many different factors shaping the bacterial community. Studies on the ecophysiology (22) and maturation of the porcine ileal microbiota (13, 27) indicate a drastic impact directly after weaning and a gradual decline of modifications during the following 2 weeks. Thus, the time point for analysis chosen in this study (14 days postweaning) does reflect a more stable period of the ileal porcine microbiota. In this study, we used bar-coded pyrosequencing of 16S rRNA genes to gain further insight into the mode of action of pharmacological levels of ZnO in the gastrointestinal tract of young pigs.Total DNA was extracted from the ileal digesta of 40- to 42-day-old piglets using a commercial kit (Qiagen stool kit; Qiagen, Hilden, Germany) and PCR amplified with unique bar-coded primer sets targeting the V1-to-V3 and the V6-to-V8 hypervariable regions (see the supplemental material for detailed methods). The rationale behind this approach was derived from the fact that no single “universal” primer pair can completely cover a complex bacterial habitat (4, 24, 32, 33). Furthermore, these studies also show that in silico information on the coverage of selected primer sets diverges from empirical results, and hence, two hypervariable regions were chosen in this study to maximize the detection of phylogenetically diverse bacterial groups.Equimolar dilutions of all samples were combined into one master sample. Pyrosequencing was performed by Agowa (Berlin, Germany) on a Roche genome sequencer FLX system using a Titanium series PicoTiterPlate. The resulting data files were uploaded to the MG-RAST server (http://metagenomics.nmpdr.org/) (19) and processed with its SEED software tool using the RDP database (5) as the reference database. After automated sequence analysis, all sequences with less than five identical reads per sample were deleted in order to increase the confidence of sequence reads and reduce bias from possible sequencing errors (12, 16). Thus, 0.43% of all sequences were not considered (1,882 of 433,302 sequences). These sequences were assigned to a total of 238 genera, of which most only occurred in a few samples (see the supplemental material). Furthermore, all unclassified sequences were removed (8.7%; 41,467 of 474,769 sequences). Due to the use of the RDP reference database, the SEED software incorrectly assigned the majority of unclassified sequences as unclassified Deferribacterales (83%; 34,393 sequences), which were actually identified as 16S soybean or wheat chloroplasts by BLAST or as cyanobacterial chloroplasts by the RDP II seqmatch tool.The pyrosequencing results for the two primer combinations were merged by taking only sequences from the primer combination that yielded the higher number of reads for a specific sequence assignment in a sample. The remaining reads were used to calculate the relative contribution of assigned sequences to total sequence reads in a sample.The Firmicutes phylum dominated the small intestinal bacterial communities in both the control group and the group with high dietary ZnO intake, with 98.3% and 97.0% of total sequence reads, respectively. No significant influence of high dietary ZnO intake was found for the main phyla Proteobacteria (0.92% versus 1.84%), Actinobacteria (0.61% versus 0.75%), Bacteroidetes (0.15% versus 0.17%), and Fusobacteria (0.09% versus 0.12%).On the order level, a total of 20 bacterial orders were detected (data not shown). Lactobacillales dominated bacterial communities in the control and high-dietary-ZnO-intake groups, with 83.37% and 93.24% of total reads. Lactic acid bacteria are well known to dominate the bacterial community in the ileum of piglets (11, 22). No significant difference between the control group and the group with high dietary ZnO intake was observed on the order level, although high dietary ZnO intake led to a strong numerical decrease for Clostridiales (14.4 ± 24.0% [mean ± standard deviation] versus 2.8 ± 1.7%), as well as to numerical increases for Pseudomonadales (0.3 ± 0.3% versus 0.6 ± 0.6%) and Enterobacteriales (0.2 ± 0.2% versus 0.5 ± 0.6%).On the genus level, a total of 103 genera were detected. Table ​Table11 summarizes the main 31 genera which exceeded 0.05% of total reads (see the supplemental material for a complete list). Lactobacilli clearly dominated the bacterial communities in both trial groups, but they also were numerically lower due to high dietary ZnO intake.

TABLE 1.

Bacterial genera in the ileum of piglets fed diets supplemented with 200 or 3,000 ppm ZnO

Growth of Arthrobacter sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen

Arthrobacter sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.Nitroglycerin (NG) is manufactured widely for use as an explosive and a pharmaceutical vasodilator. It has been found as a contaminant in soil and groundwater (7, 9). Due to NG''s health effects as well as its highly explosive nature, NG contamination in soils and groundwater poses a concern that requires remedial action (3). Natural attenuation and in situ bioremediation have been used for remediation in soils contaminated with certain other explosives (16), but the mineralization of NG in soil and groundwater has not been reported.To date, no pure cultures able to grow on NG as the sole carbon, energy, and nitrogen source have been isolated. Accashian et al. (1) observed growth associated with the degradation of NG under aerobic conditions by a mixed culture originating from activated sludge. The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3, 9, 11, 20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3, 6, 9, 11, 20), and the enzymes involved in denitration have been characterized in some detail (4, 8, 15, 21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8, 9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8, 9), and Agrobacterium radiobacter (20).Here we describe the isolation of bacteria able to degrade NG as the sole source of carbon, nitrogen, and energy. The inoculum for selective enrichment was soil historically contaminated with NG obtained at a facility that formerly manufactured explosives located in the northeastern United States. The enrichment medium consisted of minimal medium prepared as previously described (17) supplemented with NG (0.26 mM), which was synthesized as previously described (18). During enrichment, samples of the inoculum (optical density at 600 nm [OD₆₀₀] ∼ 0.03) were diluted 1/16 in fresh enrichment medium every 2 to 3 weeks. Isolates were obtained by dilution to extinction in NG-supplemented minimal medium. Cultures were grown under aerobic conditions in minimal medium at pH 7.2 and 23°C or in tryptic soy agar (TSA; 1/4 strength).Early stages of enrichment cultures required extended incubation with lag phases of over 200 h and exhibited slow degradation of NG (less than 1 μmol substrate/mg protein/h). After a number of transfers over 8 months, the degradation rates increased substantially (2.2 μmol substrate/mg protein/h). A pure culture capable of growth on NG was identified based on 16S rRNA gene analysis (504 bp) as an Arthrobacter species with 99.5% similarity to Arthrobacter pascens (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU246730","term_id":"289474572","term_text":"GU246730"}}GU246730). Purity of the cultures was confirmed microscopically and by formation of a single colony type on TSA plates. 16S gene sequencing and identification were done by MIDI Labs (Newark, DE) and SeqWright DNA Technology Services (Houston, TX). The Arthrobacter cells stained primarily as Gram-negative rods with a small number of Gram-positive cocci (data not shown); Gram variability is also a characteristic of the closely related Arthrobacter globiformis (2, 19). The optimum growth temperature is 30°C, and the optimum pH is 7.2. Higher pH values were not investigated because NG begins to undergo hydrolysis above pH 7.5 (data not shown). The isolated culture can grow on glycerol, acetate, succinate, citrate, and lactate, with nitrite as the nitrogen source. Previous authors described an Arthrobacter species able to use NG as a nitrogen source in the presence of additional sources of carbon. However, only dinitroesters were formed, and complete mineralization was not achieved (9).To determine the degradation pathway, cultures of the isolated strain (5 ml of inoculum grown on NG to an OD₆₀₀ of 0.3) were grown in minimal medium (100 ml) supplemented with NG at a final concentration of 0.27 mM. Inoculated bottles and abiotic controls were continuously mixed, and NG, 1,2DNG, 1,3DNG, 1MNG, 2MNG, nitrite, nitrate, CO₂, total protein, and optical density were measured at appropriate intervals. Nitroesters were analyzed with an Agilent high-performance liquid chromatograph (HPLC) equipped with an LC-18 column (250 by 4.6 mm, 5 μm; Supelco) and a UV detector at a wavelength of 214 nm (13). Methanol-water (50%, vol/vol) was used as the mobile phase at a flow rate of 1 ml/min. Nitrite and nitrate were analyzed with an ion chromatograph (IC) equipped with an IonPac AS14A anion-exchange column (Dionex, CA) at a flow rate of 1 ml/min. Carbon dioxide production was measured with a Micro Oxymax respirometer (Columbus Instruments, OH), and total protein was quantified using the Micro BCA protein assay kit (Pierce Biotechnology, IL) according to manufacturer''s instructions. During the degradation of NG the 1,2DNG concentration was relatively high at 46 and 72 h (Fig. ​(Fig.1).1). 1,3DNG, detected only at time zero, resulted from trace impurities in the NG stock solution. Trace amounts of 1MNG appeared transiently, and trace amounts of 2MNG accumulated and did not disappear. Traces of nitrite at time zero were from the inoculum. The concentration of NG in the abiotic control did not change during the experiment (data not shown).Open in a separate windowFIG. 1.Growth of strain JBH1 on NG. ×, NG; ▵, 1,2DNG; ⋄, 1MNG; □, 2MNG; ○, protein.Results from the experiment described above were used to calculate nitrogen and carbon mass balances (Tables ​(Tables11 and ​and2).2). Nitrogen content in protein was approximated using the formula C₅H₇O₂N (14). Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6, 9, 20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4, 8, 21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5, 12) or from oxidation of nitrite. Cultures supplemented with glycerol or other carbon sources assimilated all of the nitrite (data not shown).

TABLE 1.

Nitrogen mass balance

Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA (ssDNA) parvoviruses that belong to the Dependovirus genus and require helper viruses, such as Adenovirus or Herpesvirus, for lytic infection (4, 8, 22, 67). These viruses package a genome of ∼4.7 kb inside an icosahedral capsid (∼260 Å in diameter) with a triangulation number equal to 1 assembled from a total of 60 copies of their overlapping capsid viral protein (VP) 1 (VP1), VP2, and VP3 in a predicted ratio of 1:1:8/10 (10). The VPs are encoded from a cap open reading frame (ORF). VP3 is 61 kDa and constitutes 90% of the capsid''s protein composition. The less abundant VPs, VP1 (87 kDa) and VP2 (73 kDa), share the same C-terminal amino acid sequence with VP3 but have additional N-terminal sequences. A rep ORF codes for four overlapping proteins required for replication and DNA packaging.To date, more than 100 AAV isolates have been identified (21). Among the human and nonhuman primate AAVs isolated, 12 serotypes (AAV serotype 1 [AAV1] to AAV12) have been described and are classified into six phylogenetic clades on the basis of their VP sequences and antigenic reactivities, with AAV4 and AAV5 considered to be clonal isolates (21). AAV1 and AAV6, which represent clade A, differ by only 6 out of 736 VP1 amino acids (5 amino acids within VP3) and are antigenically cross-reactive. Other clade representatives include AAV2 (clade B), AAV2-AAV3 hybrid (clade C), AAV7 (clade D), AAV8 (clade E), and AAV9 (clade F) (21).The AAVs are under development as clinical gene delivery vectors (e.g., see references 5, 9, 12, 13, 24, 25, 53, and 61), with AAV2, the prototype member of the genus, being the most extensively studied serotype for this application. AAV2 has been successfully used to treat several disorders, but its broad tissue tropism makes it less effective for tissue-specific applications and the prevalence of preexisting neutralizing antibodies in the human population (11, 43) limits its utilization, especially when readministration is required to achieve a therapeutic outcome. Efforts have thus focused on characterizing the capsid-associated tissue tropism and transduction properties conferred by the capsid of representative serotypes of other clades (21). Outcomes of these studies include the observation that AAV1 and AAV6, for example, transduce liver, muscle, and airway epithelial cells more efficiently (e.g., up to 200-fold) than AAV2 (27, 28, 30). In addition, the six residues (Table ​(Table1)1) that differ between the VPs of AAV1 and AAV6 (a natural recombinant of AAV1 and AAV2 [56]) confer functional disparity between these two viruses. For example, AAV6 shows ∼3-fold higher lung cell epithelium transduction than AAV1 (27), and AAV1 and AAV6 bind terminally sialylated proteoglycans as their primary receptor, whereas AAV6 additionally binds to heparan sulfate (HS) proteoglycans with moderate affinity (70, 71). Therefore, a comparison of the AAV1 and AAV6 serotypes and, in particular, their capsid structures can help pinpoint the capsid regions that confer differences in cellular recognition and tissue transduction.

TABLE 1.

Amino acid differences between AAV1 and AAV6 and their reported mutants

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Use of a Mixture of Surrogates for Infectious Bioagents in a Standard Approach to Assessing Disinfection of Environmental Surfaces

We used a mixture of surrogates (Acinetobacter baumannii, Mycobacterium terrae, hepatitis A virus, and spores of Geobacillus stearothermophilus) for bioagents in a standardized approach to test environmental surface disinfectants. Each carrier containing 10 μl of mixture received 50 μl of a test chemical or saline at 22 ± 2°C. Disinfectant efficacy criteria were ≥6 log₁₀ reduction for the bacteria and the spores and ≥3 log₁₀ reduction for the virus. Peracetic acid (1,000 ppm) was effective in 5 min against the two bacteria and the spores but not against the virus. Chlorine dioxide (CD; 500 and 1,000 ppm) and domestic bleach (DB; 2,500, 3,500, and 5,000 ppm) were effective in 5 min, except for sporicidal activity, which needed 20 min of contact with either 1,000 ppm of CD or the two higher concentrations of DB.Disinfectant testing with a single type of organism does not represent field conditions, where bioagents or other pathogens may be mixed with other contaminants. Such an approach also cannot predict the true spectrum of microbicidal activity of a given chemical, while the identity of the target pathogen(s) is often unknown. We used a mixture of Acinetobacter baumannii, Mycobacterium terrae (15), hepatitis A virus (HAV) (4), and the spores of Geobacillus stearothermophilus as surrogates for infectious bioagents, with an added soil load on disks (1 cm in diameter; 0.75 mm thick) of brushed stainless steel (AISI no. 430; Muzeen & Blythe, Winnipeg, MB, Canada), to better simulate environmental surface disinfection (1, 11). Table ​Table11 gives details on the microbial strains, media used for their culture and recovery, and methods for preparing working stocks. The quantitative carrier test (QCT) method, ASTM standard E-2197 (1), was used to test the organisms singly and in a mixture. Each 200 μl of the inoculum contained 34 μl each of the four organisms, 40 μl of bovine mucin, 14 μl of yeast extract, and 10 μl of bovine serum albumin stocks.

TABLE 1.

Organisms in the mixture and their growth/recovery media and titers

A Targeted Multilocus Genotyping Assay for Lineage,Serogroup, and Epidemic Clone Typing of Listeria monocytogenes

A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table ​(Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO₄, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.

TABLE 1.

Primers used in multiplex amplification for the TMLGT assay

Quantitative Fluorescence In Situ Hybridization of Microbial Communities in the Rumens of Cattle Fed Different Diets

At present there is little quantitative information on the identity and composition of bacterial populations in the rumen microbial community. Quantitative fluorescence in situ hybridization using newly designed oligonucleotide probes was applied to identify the microbial populations in liquid and solid fractions of rumen digesta from cows fed barley silage or grass hay diets with or without flaxseed. Bacteroidetes, Firmicutes, and Proteobacteria were abundant in both fractions, constituting 31.8 to 87.3% of the total cell numbers. They belong mainly to the order Bacteroidales (0.1 to 19.2%), hybridizing with probe BAC1080; the families Lachnospiraceae (9.3 to 25.5%) and Ruminococcaceae (5.5 to 23.8%), hybridizing with LAC435 and RUM831, respectively; and the classes Deltaproteobacteria (5.8 to 28.3%) and Gammaproteobacteria (1.2 to 8.2%). All were more abundant in the rumen communities of cows fed diets containing silage (75.2 to 87.3%) than in those of cows fed diets containing hay (31.8 to 49.5%). The addition of flaxseed reduced their abundance in the rumens of cows fed silage-based diets (to 45.2 to 58.7%) but did not change markedly their abundance in the rumens of cows fed hay-based diets (31.8 to 49.5%). Fibrolytic species, including Fibrobacter succinogenes and Ruminococcus spp., and archaeal methanogens accounted for only a small proportion (0.4 to 2.1% and 0.2 to 0.6%, respectively) of total cell numbers. Depending on diet, between 37.0 and 91.6% of microbial cells specifically hybridized with the probes used in this study, allowing them to be identified in situ. The identities of other microbial populations (8.4 to 63.0%) remain unknown.The rumen is an anaerobic ecosystem used by herbivores to convert fibrous plant material into fermentation products that are in turn used as energy by the host. Fibrolytic degradation is accomplished by a complex microbial community which includes specialized fungi, protozoa, and bacteria (14). More than 200 bacterial species (5) have been isolated from rumen, and many of these have been phylogenetically and physiologically characterized. Several of these, including Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens, have the ability to hydrolyze cellulose in axenic culture (24). Despite the presence of these fibrolytic populations, a large portion of the fiber in low-quality forage diets passes through the rumen undigested. In the rumen, fibrolytic bacteria do not digest plant cell walls in isolation but rather interact with a consortium of bacteria (18). Although culture-dependent studies have improved our understanding of rumen microbiology, the importance of the isolates to the structure and function of the rumen microbial community, with the possible exception of the fibrolytic strains, is still unknown. Expanding our knowledge of the structure and function of the rumen microbial community may provide insights into approaches to improve the efficiency of fiber digestion and biofuel production (14).To provide a high-resolution view of the population structure of the rumen bacterial community, we used quantitative fluorescence in situ hybridization (qFISH) to investigate the composition and distribution of bacterial populations associated with the liquid and solid rumen contents from 12 ruminally cannulated Holstein dairy cows (3 cows were used for each diet) fed (for at least 21 days) grass hay or barley silage diets with or without flaxseed (Table ​(Table1).1). Six new 16S rRNA-targeted FISH probes (Table ​(Table2)2) for not only the fibrolytic groups but also other unclassified bacterial groups in the rumen were designed, using ARB software (17), against the rumen 16S rRNA gene sequences (data not shown) retrieved from the Ribosomal Database Project (RDP) database (6). The new probes target Bacteroidales-related clones (probe BAC1080) (phylum Bacteroidetes), Lachnospiraceae- and Ruminococcaceae-related clones (probes LAC435 and RUM831, respectively) (phylum Firmicutes), Butyrivibrio fibrisolvens-related clones (probe BFI826), and R. albus- and R. flavefaciens-related clones (probes RAL1436 and RFL155, respectively).

TABLE 1.

Composition of diets used in this study

Environmental Isolates of Burkholderia pseudomallei in Ceará State,Northeastern Brazil

Melioidosis has been considered an emerging disease in Brazil since the first cases were reported to occur in the northeast region. This study investigated two municipalities in Ceará state where melioidosis cases have been confirmed to occur. Burkholderia pseudomallei was isolated in 26 (4.3%) of 600 samples in the dry and rainy seasons.Melioidosis is an endemic disease in Southeast Asia and northern Australia (2, 4) and also occurs sporadically in other parts of the world (3, 7). Human melioidosis was reported to occur in Brazil only in 2003, when a family outbreak afflicted four sisters in the rural part of the municipality of Tejuçuoca, Ceará state (14). After this episode, there was one reported case of melioidosis in 2004 in the rural area of Banabuiú, Ceará (14). And in 2005, a case of melioidosis associated with near drowning after a car accident was confirmed to occur in Aracoiaba, Ceará (11).The goal of this study was to investigate the Tejuçuoca and Banabuiú municipalities, where human cases of melioidosis have been confirmed to occur, and to gain a better understanding of the ecology of Burkholderia pseudomallei in this region.We chose as central points of the study the residences and surrounding areas of the melioidosis patients in the rural areas of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50′W) (Fig. ​(Fig.1).1). There are two well-defined seasons in each of these locations: one rainy (running from January to May) and one dry (from June to December). A total of 600 samples were collected at five sites in Tejuçuoca (T1, T2, T3, T4, and T5) and five in Banabuiú (B1, B2, B3, B4, and B5), distributed as follows (Fig. ​(Fig.2):2): backyards (B1 and T1), places shaded by trees (B2 and T2), water courses (B3 and T3), wet places (B4 and T4), and stock breeding areas (B5 and T5).Open in a separate windowFIG. 1.Municipalities of Banabuiú (5°18′35″S, 38°55′14″W) and Tejuçuoca (03°59′20″S, 39°34′50″W).Open in a separate windowFIG. 2.Soil sampling sites in Banabuiú and Tejuçuoca.Once a month for 12 months (a complete dry/rainy cycle), five samples were gathered at five different depths: at the surface and at 10, 20, 30 and 40 cm (Table ​(Table1).1). The samples were gathered according to the method used by Inglis et al. (9). Additionally, the sample processing and B. pseudomallei identification were carried out as previously reported (1, 8, 9).

TABLE 1.

Distribution of samples with isolates by site and soil depth

Modified Bacillus thuringiensis Toxins and a Hybrid B. thuringiensis Strain Counter Greenhouse-Selected Resistance in Trichoplusia ni

Resistance of greenhouse-selected strains of the cabbage looper, Trichoplusia ni, to Bacillus thuringiensis subsp. kurstaki was countered by a hybrid strain of B. thuringiensis and genetically modified toxins Cry1AbMod and Cry1AcMod, which lack helix α-1. Resistance to Cry1AbMod and Cry1AcMod was >100-fold less than resistance to native toxins Cry1Ab and Cry1Ac.Insecticidal proteins from Bacillus thuringiensis are used widely for pest control, but evolution of resistance by pests can reduce their efficacy (3, 4, 6, 14). Resistance to B. thuringiensis toxins has been reported in field populations of four species of Lepidoptera, one species in response to sprays (3, 14) and three species in response to transgenic crops (10, 15, 16). Here, we focus on understanding and countering resistance to sprays of Bacillus thuringiensis subsp. kurstaki that evolved in commercial greenhouse populations of the cabbage looper, Trichoplusia ni (7, 17).We compared responses to single toxins and formulations of B. thuringiensis by two resistant strains (GipBtR and GlenBtR) and two related susceptible strains (GipS and GlenS) of T. ni. All four strains were started by the collection of larvae in 2001 from commercial greenhouses near Vancouver in British Columbia, Canada (7). Resistance evolved in the greenhouses in response to repeated sprays of DiPel (7), a formulation of B. thuringiensis subsp. kurstaki strain HD1 containing Cry1Aa, Cry1Ab, Cry1Ac, and Cry2Aa (9). Previously reported concentrations required to kill 50% of larvae (LC₅₀s) indicated that, relative to a susceptible laboratory strain, initial resistance to DiPel was 113-fold in the Gip population (labeled T2c in reference 7) and 24-fold in the Glen population (labeled P5 in reference 7).We reared larvae on a wheat germ diet (5) at 26°C on a light-to-dark schedule of 16 h:8 h. GipS and GlenS were reared on diet without B. thuringiensis toxins, which allowed resistance to decline (7). To maintain resistance, GipBtR and GlenBtR were reared each generation on a diet treated with 5 or 10 mg of DiPel WP (Abbott Laboratories, Ontario, Canada) per milliliter of diet (7). In bioassays, groups of five third-instar larvae were put in 60-ml plastic cups containing diet, and mortality was assessed after 3 days by gently probing larvae for movement.We used diet overlay bioassays to evaluate the toxicity to GipBtR and GipS of the protoxin forms of Cry1Ab, Cry1Ac, Cry1AbMod, and Cry1AcMod produced in B. thuringiensis strains (12). Cry1AbMod and Cry1AcMod are genetically engineered variants of Cry1Ab and Cry1Ac, respectively, each lacking 56 amino acids from the amino-terminal region, including helix α-1 (12). An 80-μl aliquot containing distilled water and toxin was dispensed evenly over the surfaces of 2 ml of diet (a mean surface area of 7.1 cm²) and allowed to dry. Fifty to 200 larvae from each strain were tested at five to eight concentrations of each toxin.We used diet incorporation bioassays (7) to evaluate the toxicities of DiPel and Agree WG (Certis, Columbia, MD) to GipS, GipBtR, GlenS, and GlenBtR. Agree is a formulation of hybrid strain GC91, which was created from the conjugation-like transfer of a plasmid from B. thuringiensis subsp. kurstaki strain HD191 into B. thuringiensis subsp. aizawai strain HD135, and it contains Cry1Ac, Cry1C, and Cry1D (1, 8). DiPel and Agree were diluted in distilled water and mixed into diet (7). Twenty-five to 50 larvae from each strain were tested at six to seven concentrations of DiPel and Agree.We used probit analysis (13) to estimate the LC₅₀s and their 95% fiducial limits (FL), as well as the slopes of concentration-mortality lines and their standard errors. The mortality of larvae fed treated diet was not adjusted for the mortality of control larvae on untreated diet, because the control mortality was low (mean, 3.6%; range, 0 to 16%). LC₅₀s with nonoverlapping 95% FL are significantly different. Resistance ratios were calculated as the LC₅₀ of a resistant strain (GipBtR or GlenBtR) divided by the LC₅₀ of its susceptible counterpart (GipS or GlenS).The genetically modified toxins Cry1AbMod and Cry1AcMod were much more effective than the native toxins Cry1Ab and Cry1Ac against larvae of T. ni from the resistant GipBtR strain (Table ​(Table1).1). Resistance ratios of GipBtR were 580 for Cry1Ab and 1,400 for Cry1Ac but only 5.5 for Cry1AbMod and 9.3 for Cry1AcMod (Table ​(Table1).1). Against GipBtR, the LC₅₀ was 53-fold higher for Cry1Ab than for Cry1AbMod and 11-fold higher for Cry1Ac than for Cry1AcMod (Table ​(Table1).1). Against GipS, however, the LC₅₀ was 2-fold higher for Cry1AbMod than for Cry1Ab and 14-fold higher for Cry1AcMod than for Cry1Ac (Table ​(Table11).

TABLE 1.

Responses of resistant (GipBtR and GlenBtR) and susceptible (GipS and GlenS) strains of T. ni to native toxins (Cry1Ab and Cry1Ac), modified toxins (Cry1AbMod and Cry1AcMod), and formulations (DiPel and Agree)

Hydroxylation and Further Oxidation of Δ9-Tetrahydrocannabinol by Alkane-Degrading Bacteria

The microbial biotransformation of Δ⁹-tetrahydrocannabinol was investigated using a collection of 206 alkane-degrading strains. Fifteen percent of these strains, mainly gram-positive strains from the genera Rhodococcus, Mycobacterium, Gordonia, and Dietzia, yielded more-polar derivatives. Eight derivatives were produced on a mg scale, isolated, and purified, and their chemical structures were elucidated with the use of liquid chromatography-mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and two-dimensional NMR (¹H-¹H correlation spectroscopy and heteronuclear multiple bond coherence). All eight biotransformation products possessed modified alkyl chains, with hydroxy, carboxy, and ester functionalities. In a number of strains, β-oxidation of the initially formed C₅ carboxylic acid led to the formation of a carboxylic acid lacking two methylene groups.Δ⁹-Tetrahydrocannabinol (Δ⁹-THC) is the decarboxylated product of the corresponding Δ⁹-THC acid, the major cannabinoid present in the cannabis plant (Cannabis sativa L., Cannabaceae). This compound is officially registered as a drug for the stimulation of appetite and antiemesis in patients under chemotherapy and human immunodeficiency virus therapy regimens. Other biological activities ascribed to this compound include lowering intraocular pressure in glaucoma, acting as an analgesic for muscle relaxation, immunosuppression, sedation, bronchodilation, and neuroprotection (11).Δ⁹-THC and many of its derivatives are highly lipophilic and poorly water soluble. Calculations of the n-octanol/water partition coefficient (K_o/w) of Δ⁹-THC at neutral pH vary between 6,000, using the shake flask method (15), and 9.44 × 10⁶, by reverse-phase high-performance liquid chromatography estimation (19). The poor water solubility and high lipophilicity of cannabinoids cause their absorption across the lipid bilayer membranes and fast elimination from blood circulation. In terms of the “Lipinsky rule of 5” (14), the high lipophilicity of cannabinoids hinders the further development of these compounds into large-scale pharmaceutical products.To generate more water-soluble analogues, one can either apply de novo chemical synthesis (as, e.g., in reference 16) or modify naturally occurring cannabinoids, e.g., by introducing hydroxy, carbonyl, or carboxy groups. Chemical hydroxylation of compounds such as cannabinoids is difficult (Δ⁹-THC is easily converted into Δ⁸-THC under mild conditions), and therefore microbial biotransformation of cannabinoids is potentially a more fruitful option to achieve this goal.So far, studies on biotransformation of Δ⁹-THC were mainly focused on fungi, which led to the formation of a number of mono- and dihydroxylated derivatives. Previous reports on the biotransformation of cannabinoids by various microorganisms are summarized in Table ​Table1.1. The aim of the present study was to test whether bacterial strains are capable of transforming Δ⁹-THC into new products (with potentially better pharmaceutical characteristics) at a higher yield and specificity than previously found for fungal strains. For this purpose, we have chosen to use a collection of alkane-degrading strains, since it was shown in previous studies (8, 18, 20) that alkane oxygenases often display a broad substrate range. Production of novel cannabinoid derivatives that might have interesting pharmacological activities was another objective of this project.

TABLE 1.

Previous biotransformation experiments conducted using various microorganisms to transform cannabinoids

Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity,Resistance to Hepatic Steatosis,and Defective Gluconeogenesis

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a^−/− but not in control or Par-1b^−/− mice. The intercrossing of Par-1a^−/− with Par-1b^−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a^−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b^−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.Cellular polarity is a fundamental principle in biology (6, 36, 62). The prototypical protein kinase originally identified as a regulator of polarity was termed partitioning defective (Par-1) due to early embryonic defects in Caenorhabditis elegans (52). Subsequent studies revealed that Par-1 is required for cellular polarity in worms, flies, frogs, and mammals (4, 17, 58, 63, 65, 71, 89). An integral role for Par-1 kinases in multiple signaling pathways has also been established, and although not formally addressed, multifunctionality for individual Par-1 family members is implied in reviews of the list of recognized upstream regulators and downstream substrates (Table ​(Table1).1). Interestingly, for many Par-1 substrates the phosphorylated residues generate 14-3-3 binding sites (25, 28, 37, 50, 59, 61, 68, 69, 78, 95, 101, 103). 14-3-3 binding in turn modulates both nuclear/cytoplasmic as well as cytoplasmic/membrane shuttling of target proteins, thus allowing Par-1 activity to establish intracellular spatial organization (15, 101). The phosphorylation of Par-1 itself promotes 14-3-3 binding, thereby regulating its subcellular localization (37, 59, 101).

TABLE 1.

Multifunctionality of Par-1 polarity kinase pathways^a