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

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus

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

Frequency and Rate of Pilin Antigenic Variation of Neisseria meningitidis

The rates of pilin antigenic variation (Av) of two strains of Neisseria meningitidis were determined using an unbiased DNA sequencing assay. Strain MC58 underwent pilin Av at a rate similar to that of N. gonorrhoeae strain MS11 but lower than that of N. gonorrhoeae strain FA1090. Pilin Av was undetectable in strain FAM18.Neisseria meningitidis is a Gram-negative diplococcus that colonizes the nasopharynx of approximately 5 to 10% of the population and is usually nonpathogenic but can occasionally enter the bloodstream to cause septicemia and can eventually spread to the meninges, causing meningitis (15). Approximately 500,000 cases of meningococcal meningitis occur every year, with nearly 10% resulting in fatality (2).Type IV pili (TFP) are long filamentous structures protruding from the bacterial surface and are required for adherence of N. meningitidis to host cells (7). As with the TFP of the closely related pathogen Neisseria gonorrhoeae, the pili are able to undergo antigenic variation (Av). In N. gonorrhoeae, pilin Av occurs as a result of recombination between one of the multiple silent pilS copies and the expressed pilin gene (pilE). The pilS copies share significant regions of homology with pilE yet lack a promoter or ribosome-binding site and the initial 5′ coding sequence. Pilin Av relies on RecA and the RecF-like recombination pathway to catalyze gene conversion, resulting in an altered pilE sequence, carrying part of the pilS donor, and the original unaltered pilS sequence (8, 9).While the frequency of pilin Av has been measured in N. gonorrhoeae (5, 10, 12), this process has never been quantified in N. meningitidis. Two sequenced strains were picked to measure pilin Av: serogroup B strain MC58 (sequence type [ST-32] complex), isolated from an invasive infection (14), and serogroup C strain FAM18 (ST-11 complex), which was isolated from a patient with septicemia (1). In both strains, the native recA gene was replaced with the very highly conserved N. gonorrhoeae recA6 construct, which allows regulation of expression with IPTG (isopropyl-β-d-thiogalactopyranoside) (17). recA6 strains are RecA⁺ when grown with IPTG but are RecA⁻ when grown without IPTG (17). These phenotypes were confirmed by measuring the UV sensitivities and DNA transformation competence levels of both strains with or without IPTG, and both strains were shown to be piliated by transmission electron microscopy (data not shown). Bacteria were grown at 37°C with 5% CO₂ on gonococcal medium base (GCB; Difco) plus Kellogg supplements I and II (11).The pilin Av sequencing assay was performed as described previously (5, 10, 12) with slight modifications. Briefly, FAM18 and MC58 were grown on solid GCB with 1 mM IPTG, allowing for the expression of RecA, for 22 h and 12.5 h, respectively, which was estimated to produce 20 generations. For FAM18, little or no pilin Av was expected since the G-quartet-forming sequence required for pilin Av is degenerate in this strain (3). Therefore, two random progenitor colonies were picked from IPTG-enriched medium and passaged on GCB without IPTG. Between 91 and 94 colonies were isolated from each FAM18 progenitor, and the sequence of the pilE gene was determined. For MC58, seven random progenitor colonies were picked and passaged on GCB without IPTG. Between 28 and 47 progeny colonies arising from each of the seven progenitors were isolated, and the pilE gene sequence was determined. In both MC58 and FAM18, the progeny colonies were passaged on GCB two times to ensure colony clonality. A single colony from each sample was isolated, and the pilE gene was PCR amplified as described previously (13).The primers used for amplification of MC58 pilE were McPilRBS (5′-GCATTTCCTTTCCAATTAGGAG) and MC58SP3A (5′-TTCCGTACGGATAGCTTCGTC). The primers used for amplification of FAM18 pilE were FAMFOR-2 (5′-ATTACGGGTTTACGTTTGCGG) and FAMREV-2 (5′-ACGCACCTACGCCTCACCCTAC). The DNA sequence was determined for each sample (SeqWright, Houston, TX, and the Genomics Core at Northwestern University) and analyzed (MacVector; Symantec Corp.). Colonies that showed pilE sequence changes were reanalyzed to confirm the Av event.The pilin Av frequency was determined for the progeny of each progenitor by dividing the total number of detected pilin Av events by the number of progeny of each set, resulting in two values for FAM18 and seven values for MC58 (Table ​(Table1).1). The pilin Av rate was determined by dividing the pilin Av frequency by the number of generations for each sample grown in the presence of IPTG, as determined by a colony assay at the time of harvest. After growth for the same time period, the total numbers of generations for MC58 and FAM18 grown under RecA induction were approximately 19 and 23, respectively.

TABLE 1.

Frequencies and rates of pilin Av in MC58 and FAM18^a

Elimination of d-Lactate Synthesis Increases Poly(3-Hydroxybutyrate) and Ethanol Synthesis from Glycerol and Affects Cofactor Distribution in Recombinant Escherichia coli

The effect of eliminating d-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP⁺ ratio was higher than the NADH/NAD⁺ ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP⁺. In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.Poly(3-hydroxybutyrate) (PHB) is the best-known and most common polyhydroxyalkanoate (PHA). PHAs are polymers with thermoplastic properties that are totally biodegradable by microorganisms present in most environments and that can be produced from different renewable carbon sources (38). Accumulated as intracellular granules by many bacteria under unfavorable conditions (1, 21), PHAs are carbon and energy reserves and also act as electron sinks, enhancing the fitness and stress resistance of bacteria and contributing to redox balance (12, 30). Escherichia coli offers a well-defined physiological environment for the construction and manipulation of various metabolic pathways to produce different bioproducts, such as PHB, from cost-effective carbon sources.In recent years, a significant increase in the production of biodiesel has caused a sharp fall in the cost of glycerol, the main by-product of biodiesel synthesis. As a result, glycerol has become a very attractive substrate for bacterial fermentations (10), specially for reduced products, such as PHB (36). The E. coli strain used in this work, K24K, carries phaBAC, the structural genes responsible for PHB synthesis, from Azotobacter sp. strain FA8 (23) (Table ​(Table1).1). The pha genes in K24K are expressed from a chimeric promoter and consequently are not subject to the genetic regulatory systems present in natural PHA producers. Because of this, it can be assumed that regulation of PHA synthesis in the recombinants is restricted by enzyme activity levels, modulated principally by substrate availability. In most natural producers, and also in PHB-producing E. coli recombinants, PHB is synthesized through the condensation of two molecules of acetyl-coenzyme A (acetyl-CoA), catalyzed by an acetoacetyl-CoA transferase or 3-ketothiolase, resulting in acetoacetyl-CoA. This compound is subsequently reduced by an NAD(P)H-dependent acetoacetyl-CoA reductase to R-(−)-3-hydroxybutyryl-CoA, which is then polymerized by a specific PHA synthase (34).

TABLE 1.

E. coli strains, plasmids, and oligonucleotides used in this study

Identification of the Site-Specific DNA Invertase Responsible for the Phase Variation of SusC/SusD Family Outer Membrane Proteins in Bacteroides fragilis

The human gut microbe Bacteroides fragilis can alter the expression of its surface molecules, such as capsular polysaccharides and SusC/SusD family outer membrane proteins, through reversible DNA inversions. We demonstrate here that DNA inversions at 12 invertible regions, including three gene clusters for SusC/SusD family proteins, were controlled by a single tyrosine site-specific recombinase (Tsr0667) encoded by BF0667 in B. fragilis strain YCH46. Genetic disruption of BF0667 diminished or attenuated shufflon-type DNA inversions at all three susC/susD genes clusters, as well as simple DNA inversions at nine other loci, most of which colocalized with susC/susD family genes. The inverted repeat sequences found within the Tsr0667-regulated invertible regions shared the consensus motif sequence AGTYYYN₄GDACT. Tsr0667 specifically mediated the DNA inversions of 10 of the 12 regions, even under an Escherichia coli background when the invertible regions were exposed to BF0667 in E. coli cells. Thus, Tsr0667 is an additional globally acting DNA invertase in B. fragilis, which probably involves the selective expression of SusC/SusD family outer membrane proteins.The human gut harbors an abundant and diverse microbiota. Bacteroides is one of the most abundant genera of human gut microflora (10, 17, 20), and the biological activities of Bacteroides species are deeply integrated into human physiology through nutrient degradation, the production of short-chain fatty acids, or immunomodulatory molecules (11-14, 24). Recent genomic analyses of Bacteroides have revealed that the bacteria possess redundant abilities not only to bind and degrade otherwise indigestible dietary polysaccharides but also to produce vast arrays of capsular polysaccharide (5, 19, 38, 39). These functional redundancies have been established by the extensive duplication of various genes that encode molecules such as glycosylhydrolases, glycosyltransferases, and outer membrane proteins of the SusC/SusD family (starch utilization system) known to be involved in polysaccharide recognition and transport (7, 27, 28, 30). It has been assumed that these functional redundancies of Bacteroides contribute to the stability of the gut ecosystem (3, 21, 23, 32, 39).Another characteristic feature common in Bacteroides species is that the expression of some of the genes is altered in an on-off manner by reversible DNA inversions at gene promoters or within the protein-coding regions (5, 9, 19, 38, 39). These phase-variable phenotypes are associated with surface architectures such as capsular polysaccharides and SusC/SusD family proteins (5, 6, 16, 19). Our previous genomic analyses of Bacteroides fragilis strain YCH46 revealed that it contained as many as 31 invertible regions in its chromosome (19). These invertible regions can be grouped into six classes according to the internal motif sequences within inverted repeat sequences (IRs) (Table ​(Table1).1). The DNA inversions within these regions are thought to be controlled by site-specific DNA invertases specific to each class. B. fragilis strain YCH46 contains 33 tyrosine site-specific recombinases (Tsr) genes and four serine site-specific recombinases (Ssr) genes. Generally, DNA invertases mediate DNA inversions at adjacent regions, such as FimB and FimE, that flip their immediate downstream promoters to generate a phase-variable phenotype of type I pili in Escherichia coli (15). B. fragilis is unique in that this anaerobe possesses not only locally acting DNA invertases but also globally acting DNA invertases that mediate DNA inversions at distant loci (8, 29). It has been reported that B. fragilis possesses at least two types of master DNA invertase that regulate DNA inversions at multiple loci simultaneously (8, 29). One is Mpi, an Ssr that mediates the on-off switching of 13 promoter regions (corresponding to class I regions in B. fragilis strain YCH46), including seven promoter regions for capsular polysaccharide biosynthesis in B. fragilis strain NCTC9343 (8). The other master DNA invertase is Tsr19, a Tsr that regulates DNA inversions at two distantly located promoter regions (corresponding to class IV regions in B. fragilis strain YCH46) associated with the large encapsulation phenotype (6, 26, 29). The invertible regions contain specific consensus motifs within the IRs corresponding to each DNA invertase and constitute a regulatory unit. We designated this type of regulatory unit as an “inverlon,” which consists of at least two invertible regions controlled by a single master DNA invertase.

TABLE 1.

Classification of the invertible regions in B. fragilis strain YCH46 based on internal motif sequences within IRs

Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle

The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.The gastrointestinal tracts (GIT) of beef cattle are colonized by microorganisms that profoundly impact animal physiology, nutrition, health, and productivity (5). The GIT microbiota potentially impact food safety via pathogen shedding (13) by interacting with organisms such as Salmonella and competing for resources in the GIT. Cattle intestinal microbiota also play an important role in methane emissions, with U.S. beef cattle alone contributing an estimated 3.87 million metric tons of methane into the environment each year, both from rumen and large-intestine fermentations (7). Although the bovine fecal microbiota have been well characterized using culture-based methods, these techniques are necessarily limited to characterizing bacteria that can be grown in the laboratory. Culture-independent methods can reveal community members that are recalcitrant to culture. Only a handful of deep-sequencing studies have been done using culture-independent 16S rRNA-based methods (1, 11, 12, 14), all with dairy cattle, which have a fundamentally different diet and metabolism from beef cattle. Despite the potential contributions of the beef cattle GIT microbiota to animal health, food safety, and global warming, these communities remain poorly characterized. With the advent of pyrosequencing technology, researchers now have the tools to characterize these important communities. Pyrosequencing will allow rapid characterization of large-sample data sets (1). However, the taxonomic information generated by rapid sequencing is approximate by necessity (9), and full-length 16S-rRNA sequencing remains the “gold standard” method. Accordingly, we have characterized fecal bacteria from six feedlot cattle by full-length capillary sequence analysis of 11,171 16S rRNA gene clones (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Bacterial diversity of six feedlot beef cattle. Gray bars represent the percentages of all 16S sequences that were assigned to each taxonomy. Colored dots represent the percentages of 16S sequences from each library that were assigned to each taxonomic group. Asterisks indicate unclassified members of the named taxon. Panel A shows the data for the first 99% of all the sequences. Panel B shows the data for the remaining 1% of sequences. Note differences in scales for panels A and B.Rectal grab fecal samples (n = 6) were collected according to institutional animal care guidelines. All animals were female cross-bred MARCIII beef heifers, 6 to 8 months of age, 214 to 241 kg, housed in the same feedlot pen for 2 months prior to fecal collection, and fed the same typical feedlot beef production growing rations consisting of 61.6% corn silage (41.3% dry matter), 15.2% alfalfa hay, 20.9% corn, and 2.3% liquid supplement.Total fecal DNA was isolated from homogenized samples using MoBio UltraClean fecal kit (Carlsbad, CA). PCR was performed using 27F and 1392R primers (11). Amplification consisted of 25 cycles, with an annealing temperature of 55°C. Amplicons from three reactions per sample were pooled (8), cloned using the Invitrogen TOPO TA cloning kit (Carlsbad, CA), and sequenced bidirectionally with M13 primers using an ABI 3700 sequencer (17). Low-quality and chimeric sequences (6) were excluded from further analysis. Distance matrices were compiled from ClustalW alignments (18) in PHYLIP (4). Pairwise estimates of shared richness were calculated using EstimateS, version 8.2 (R. K. Colwell; http://purl.oclc.org/estimates). DOTUR (16) was used to identify operational taxonomic units (OTUs) and to generate rarefaction curves (Fig. ​(Fig.2),2), richness and evenness estimates, and Shannon''s and Simpson''s diversity indices (Table ​(Table1).1). A 97% similarity cutoff and an 85% similarity cutoff for estimating OTUs were used to approximate species and class-level designations (15). Taxonomies were assigned to one member of each OTU using the RDP “classifier” tool (19), and the RDP taxonomic information was used for Fig. ​Fig.11 and ​and3.3. Common bovine taxa were identified based on inclusion in all three U.S. culture-independent studies (this study and references 1 and 11).Open in a separate windowFIG. 2.Rarefaction curves for six feedlot beef cattle. OTUs were assigned at the 85% DNA sequence similarity level. For comparison purposes, all six curves were truncated after 1,321 sequences.Open in a separate windowFIG. 3.Phylum-level distribution of bacterial sequences from six beef feedlot cattle. Asterisks indicate unclassified members of the named taxon.

TABLE 1.

Richness and diversity indices for 6 beef feedlot cattle

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

Human Bocavirus Capsid Structure: Insights into the Structural Repertoire of the Parvoviridae

Human bocavirus (HBoV) was recently discovered and classified in the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae) on the basis of genomic similarity to bovine parvovirus and canine minute virus. HBoV has been implicated in respiratory tract infections and gastroenteric disease in children worldwide, yet despite numerous epidemiological reports, there has been limited biochemical and molecular characterization of the virus. Reported here is the three-dimensional structure of recombinant HBoV capsids, assembled from viral protein 2 (VP2), at 7.9-Å resolution as determined by cryo-electron microscopy and image reconstruction. A pseudo-atomic model of HBoV VP2 was derived from sequence alignment analysis and knowledge of the crystal structure of human parvovirus B19 (genus Erythrovirus). Comparison of the HBoV capsid structure to that of parvoviruses from five separate genera demonstrates strong conservation of a β-barrel core domain and an α-helix, from which emanate several loops of various lengths and conformations, yielding a unique surface topology that differs from the three already described for this family. The highly conserved core is consistent with observations for other single-stranded DNA viruses, and variable surface loops have been shown to confer the host-specific tropism and the diverse antigenic properties of this family.Human bocavirus (HBoV), a newly discovered member of the family Parvoviridae, was originally isolated in randomly selected nasopharyngeal aspirates (5). Since this initial discovery, HBoV has also been detected worldwide, predominantly in children under the age of 2 years with respiratory infections, in serum, urine, and fecal samples (40). Symptomatic children commonly exhibit acute diseases of the upper and lower respiratory tracts (7, 36, 44, 56) and, possibly, gastroenteritis (31, 56) though the link to gastroenteritis outbreaks has been questioned (12). It is still unclear if HBoV is the sole etiologic agent of respiratory disease as higher rates of coinfections with other respiratory pathogens such as human rhinovirus and Streptococcus spp. are often observed (4). However, Allander et al. recently reported (4) that HBoV was found in 19% of children with acute wheezing, thereby making it the fourth most common virus, after rhinoviruses, enteroviruses, and respiratory syncytial virus, detected in children exhibiting this symptom. These findings suggest that, at high viral load, HBoV could be an etiologic agent of respiratory tract disease (4). HBoV infection is common in the first few years of life, and clinical research suggests it may follow the primary period for acquisition of human parvovirus B19 (B19) though there is no antigenic cross-reactivity between B19 and HBoV (28, 30). By age 5, most people have circulating antibodies against HBoV, as is also true for other respiratory viruses such as respiratory syncytial virus, rhinoviruses, and human metapneumovirus (17). HBoV has also been identified in adults, with ∼63% of samples tested being seropositive, showing a positive correlation with age and a slight positive bias toward women (14).The Parvoviridae is a family of small, nonenveloped viruses that package a single-stranded DNA (ssDNA) genome of ∼5,000 bases. These viruses are subdivided into two subfamilies: Parvovirinae and Densovirinae (Table ​(Table1).1). The Parvovirinae are further subdivided into five genera, all of whose members infect vertebrates. The Densovirinae (four genera) infect only invertebrates. Phylogenetic analysis places HBoV in the recently classified Bocavirus genus (Table ​(Table1).1). In addition to HBoV, numerous parvoviruses circulate among the human population. Among these are the following: several dependoviruses; adeno-associated virus (AAV) serotypes AAV1 to AAV3, AAV5, and AAV9; the Erythrovirus B19; and the newly discovered human parvovirus genotypes 4 (Parv4) and 5 (Parv5) (23, 27, 50). Of these, only B19 had been implicated in disease until the discovery of HBoV and Parv4, which has been isolated from patients who present symptoms of acute HIV infection (50).

TABLE 1.

Selected properties of representative members of the Parvoviridae

Selected Pseudomonas putida Strains Able To Grow in the Presence of High Butanol Concentrations

Adapted Pseudomonas putida strains grew in the presence of up to 6% (vol/vol) butanol, the highest reported butanol concentration tolerated by a microbe. P. putida might be an alternative host for biobutanol production, overcoming the primary limitation of currently used strains—insufficient product titers due to low butanol tolerance.The focus of biofuel production research has recently shifted from ethanol to bioenergy carriers that are more compatible with existing infrastructure (e.g., refineries, transport, and car engines). At the forefront is n-butanol (hereafter referred to as butanol) for which large-scale production processes have been implemented (16, 35). Existing fermentations, however, are limited in energetically attractive butanol titers, because butanol inhibits microbial growth at concentrations above 16 g/liter (2, 10). As reported for other organic solvents with low logarithm of the partition coefficient in a two-phase octanol/water system (log P_ow), this toxicity is due primarily to accumulation of butanol (log P_ow, 0.8) in the cell membrane and subsequent impairment (4, 17, 30, 33). With the maximum aqueous solubility of 0.97 M (8.8% [vol/vol]), the maximum membrane concentration of butanol was calculated to be 1.59 M (17), spotlighting its potential toxicity. The low achievable butanol titers have necessitated large reactor volumes, resulting in high purification costs (8, 15). Recent metabolic engineering strategies for improving biobutanol fermentation have focused on maximization of butanol production rates (10, 19), reducing the levels of by-products (20), finding alternative substrates (20), or finding alternative hosts (2, 12, 21, 31). However, recently engineered microbial strains (1, 14) have not overcome butanol toxicity.High organic solvent concentrations are tolerated by strains of the bacterial species Pseudomonas putida reported to grow in a second phase of octanol (25), toluene (13), or styrene (32). This suggests that solvent-tolerant P. putida strains withstand high butanol titers and therefore warrant exploitation as host for butanol production. Indeed, viable solvent-tolerant P. putida S12 cells were observed at butanol concentrations of up to 10% (vol/vol) by live-dead staining and fluorescence microscopy (5) (see supplemental material). We used growth as the parameter of interest, because growth in the presence of butanol directly indicates the potential of selected P. putida strains as hosts for recombinant butanol production.Three solvent-tolerant P. putida strains, DOT-T1E (23), S12 (32), and Pseudomonas sp. strain VLB120 (18), and the solvent-sensitive P. putida reference strain KT2440 (24) were examined for their ability to grow in the presence of butanol. Toxicity assays were performed in 96-well microtiter plates (System Duetz [7]) at 30°C and 300 rpm using glucose-supplemented LB and M9 media (with 10 and 5 g/liter glucose, respectively) (26). Higher glucose concentrations in LB medium did not increase butanol tolerance (data not shown). Butanol was added in all experiments to cells in the mid-exponential phase. Cell growth was monitored by changes in optical density, and substrate and butanol concentrations were analyzed by high-pressure liquid chromatography (Trentec 308R-Gel.H; VWR Hitachi). Comparable low butanol concentrations were withstood by all P. putida strains, with butanol tolerance highly dependent on the medium composition (Table ​(Table1).1). Growth was observed at butanol concentrations up to 3% (vol/vol), occurring in a culture of Pseudomonas sp. strain VLB120 using glucose-supplemented LB medium.

TABLE 1.

Tolerated butanol concentrations in different growth media