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

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

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

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

Wide Variation in the Multiplicity of HIV-1 Infection among Injection Drug Users

Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (14-16, 31, 34, 45). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (18-20, 43) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ∼80% of HIV-infected heterosexuals and in ∼60% of HIV-infected men who have sex with men (MSM) (1, 13, 18, 24). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (6, 11-13, 17, 25, 28, 30, 35, 38, 42, 47-49) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (15, 36).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (2, 3, 29, 42) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (26, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from single or multiple transmitted/founder viruses could be identified following intravenous inoculation and that the rectal mucosal barrier to infection was 2,000- to 20,000-fold greater than with intravenous inoculation (19). However, we also recognized potentially important differences between virus transmission in Indian rhesus macaques and virus transmission in humans that could complicate an IDU acquisition study. For example, in the SIV macaque model, the virus inocula can be well characterized genetically and the route and timing of virus exposure in relation to plasma sampling precisely defined, whereas in IDUs, the virus inoculum is generally undefined and the timing of virus infection only approximated based on clinical history and seroconversion testing (8). In addition, IDUs may have additional routes of potential virus acquisition due to concomitant sexual activity. Finally, there is a paucity of IDU cohorts for whom incident infection is monitored sufficiently frequently and clinical samples are collected often enough to allow for the identification and enumeration of transmitted/founder viruses. To address these special challenges, we proposed a pilot study of 10 IDU subjects designed to determine with 95% confidence if the proportion of multivariant transmissions in IDUs was more than 2-fold greater than the 20% frequency established for heterosexual transmission (1, 13, 18, 24). A secondary objective of the study was to determine whether the range in numbers of transmitted/founder viruses in IDUs exceeded the 1-to-6 range observed in heterosexuals (1, 13, 18, 24). To ensure comparability among the studies, we employed SGA-direct amplicon sequencing approaches, statistical methods, and power calculations identical to those that we had used previously to enumerate transmitted/founder viruses in heterosexual and MSM cohorts (1, 13, 18, 20, 24).We first surveyed investigators representing acute-infection cohorts in the United States, Canada, Russia, and China; only one cohort—the Montreal Primary HIV Infection Cohort (41)—had IDU clinical samples and clinical data available for study. The Montreal cohort of subjects with acute and early-stage HIV-1 infection was established in 1996 and recruits subjects from both academic and private medical centers throughout the city. Injection drug use is an important contributing factor to Montreal''s HIV burden, with IDUs comprising approximately 20% of the city''s AIDS cases and 35% of the cohort (21, 40, 41). A large proportion of Montreal''s IDUs use injection cocaine, with 50 to 69% of subjects reporting cocaine as their injection drug of choice (4, 5, 9, 22, 23).Subjects with documented serological evidence of recent HIV-1 infection and a concurrent history of injection drug use were selected for study. These individuals had few or no reported risk factors for sexual HIV-1 acquisition. Clinical history and laboratory tests of HIV-1 viremia and antibody seroconversion were used to determine the Fiebig clinical stage (8) and to estimate the date of infection (Table ​(Table1).1). One subject was determined to be in Fiebig stage III, one subject was in Fiebig stage IV, five subjects were in Fiebig stage V, and three subjects were in Fiebig stage VI. We performed SGA-direct amplicon sequencing on stored plasma samples and obtained a total of 391 3′ half-genomes (median, 25 per subject; range, 19 to 167). Nine of these sequences contained large deletions or were G-to-A hypermutated and were excluded from subsequent analysis. Sequences were aligned, visually inspected using the Highlighter tool (www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), and analyzed by neighbor-joining (NJ) phylogenetic-tree construction. A composite NJ tree of full-length gp160 env sequences from all 10 subjects (Fig. ​(Fig.1A)1A) revealed distinct patient-specific monophyletic lineages, each with high bootstrap support and separated from the others by a mean genetic distance of 10.79% (median, 11.29%; range, 3.00 to 13.42%). Maximum within-patient env gene diversity ranged from 0.23% to 3.34% (Table ​(Table1).1). Four subjects displayed distinctly lower within-patient maximum env diversities (0.23 to 0.49%) than the other six subjects (1.48% to 3.34%). The lower maximum env diversities in the former group are consistent with infection either by a single virus or by multiple closely related viruses, while the higher diversities can be explained only by transmission of more than one virus based on empirical observations (1, 13, 18, 24) and mathematical modeling (18, 20).Open in a separate windowFIG. 1.NJ trees and Highlighter plots of HIV-1 gp160 env sequences. (A) Composite tree of 382 gp160 env sequences from all study subjects. The numerals at the nodes indicate bootstrap values for which statistical support exceeded 70%. (B) Subject ACT54869022 sequences suggest productive infection by a single virus (V1). (C) Subject HDNDRPI032 sequences suggest productive infection by as many as three viruses. (D) Subject HDNDRPI001 sequences suggest productive infection by at least five viruses with extensive interlineage recombination. Sequences are color coded to indicate viral progeny from distinct transmitted/founder viruses. Recombinant virus sequences are depicted in black. Methods for SGA, sequencing, model analysis, Highlighter plotting, and identification of transmitted/founder virus lineages are described elsewhere (18, 20, 24, 44). The horizontal scale bars represent genetic distance. nt, nucleotide.

TABLE 1.

Subject demographics and HIV-1 envelope analysis results

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

Different Rotavirus Strains Enter MA104 Cells through Different Endocytic Pathways: the Role of Clathrin-Mediated Endocytosis

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins α2β1 and αvβ3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.Endocytosis is a cellular process that involves the formation of a vesicle whose cargo is transported from the extracellular milieu to the interior of the cell. Several endocytic pathways have been described, and all of them have been shown to be used by viruses during cell entry. These pathways include clathrin-mediated endocytosis, uptake via caveolae, macropinocytosis, phagocytosis, and a novel non-clathrin-, non-caveola-mediated pathway that is currently not well characterized (32). While detailed information about the entry of several enveloped viruses is now available (4, 35, 49, 53, 56), the mechanism by which nonenveloped viruses enter cells is not well understood. Two general mechanisms have been proposed to be used by these viruses to reach the cell''s cytoplasm: direct penetration at the cell surface, during which the viral particles are directly translocated from the external milieu into the cytoplasm, or internalization through endocytic processes (55).Rotaviruses, members of the family Reoviridae, are the leading etiologic agent of viral gastroenteritis in infants and young children worldwide, being responsible for an estimated 500,000 deaths each year (41). These nonenveloped viruses are formed by three concentric layers of protein that surround the viral genome, formed by 11 segments of double-stranded RNA. The outermost layer of the virion is formed by two proteins, VP4 and VP7, which are involved in the early interactions of the virus with its host cell (7, 27). VP4 is involved in receptor binding and cell penetration. The role of VP7 is less clear, although it has been shown that it interacts with the cell surface molecules at a postattachment step (17). After binding to the cell surface, the virus penetrates the plasma membrane to productively infect the cell. This penetration depends on the trypsin treatment of the virus, which results in the specific cleavage of VP4 to polypeptides VP8 and VP5. This cleavage promotes VP4 rearrangements in the viral particles that rigidify the spikes (7, 11).Despite the fact that, in vivo, rotaviruses primarily infect the mature enterocytes of the small intestine, studies of the infection of this type of cells have been limited due to the lack of established intestinal cell lines of small intestine origin. Given the absence of a better model, most of the studies on the entry and replication cycle of rotavirus have been conducted either in the epithelial monkey kidney cell line MA104 or in the human colon carcinoma cell line Caco-2, which are highly permissible to these viruses.Using as a model MA104 cells and the simian rotavirus RRV, we have proposed that rotavirus cell entry is a complex multistep process that involves the two virus surface proteins and several cell receptors, including sialic acids, gangliosides, integrins α2β1, α4β1, αvβ3, and αxβ2, and the heat shock cognate protein hsc70 (22). We have also shown that depletion of cholesterol from the cellular membrane severely impairs the infectivity of rotavirus (19, 50) and have suggested that sphingolipid- and cholesterol-enriched membrane lipid microdomains might be involved in rotavirus cell entry, since the virus and its receptors associate with these domains at early times during infection (23). However, there are some rotavirus strains that may not use all these molecules; some rotavirus strains are resistant to the neuraminidase (NA) treatment of the cell, and thus, they have been classified as NA resistant (6, 34). Additionally, the infectivity of some viral strains is not blocked by anti-integrin antibodies, suggesting the existence of rotavirus strains that are integrin independent (Table ​(Table1)1) (17).

TABLE 1.

Cellular receptor requirements of different rotavirus strains

Identification and Characterization of Class 1 Integron Resistance Gene Cassettes among Salmonella Strains Isolated from Imported Seafood

A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.Salmonella spp. are recognized as major food-borne pathogens of humans worldwide. In the United States, there are an estimated 800,000 to 4 million Salmonella infections annually, and approximately 500 of the cases are fatal (8, 26). A variety of foods have been implicated as vehicles transmitting salmonellosis to humans, including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fruits, juice, and vegetables (1, 4, 9, 12, 23). Previous studies by field laboratories of the U.S. Food and Drug Administration have shown the prevalences of Salmonella isolates in imported and domestic seafood as 7.2% and 1.3%, respectively (6, 11, 27).Mobile genetic elements, such as plasmids, transposons, and integrons, which disseminate antibiotic resistance genes by horizontal or vertical transfer, as part of either resistance plasmids or conjugative transposons, play an important role in the evolution and dissemination of multidrug resistance (2, 3, 10, 17). Salmonella genomic island 1 (SGI1), the first genomic island reported to contain an antibiotic resistance gene cluster, was identified in the multidrug-resistant Salmonella enterica serovar Typhimurium strain DT 104 (21).Most studies of the prevalence and characterization of antimicrobial resistance genes and integrons in Salmonella spp. have focused on strains from clinical and veterinary sources. However, little is known about the occurrence of SGI1 and its variants in Salmonella spp. isolated from seafood. We have screened a set of drug-resistant S. enterica strains from seafood belonging to 64 different serovars for SGI1 and class 1 integron conserved sequences (CS). We report the presence of a class I variant integron carrying the dfrXII and aadA2 genes on a megaplasmid in serovar Typhimurium var. Copenhagen and on the chromosome in Salmonella enterica serovar Lansing. We also found the variant class 1 integron carrying the dfrA1 and orfC genes in Salmonella enterica serovar Newport strains from seafood.A total of 210 Salmonella enterica strains isolated from seafood imported into the United States between 2000 and 2005 were identified and serotyped by the Pacific Regional Laboratory-Southwest of the FDA, Irvine, CA. The Salmonella strains represented 20 serogroups (Table ​(Table1)1) from various imported seafood items. The Salmonella strains were tested with 16 antibiotics (14) commonly used in either human or veterinary medicine on Mueller-Hinton agar (Difco Laboratories, Detroit, MI), using a disk diffusion method. The sensitivity and resistance were determined by the criteria of the Clinical and Laboratory Standards Institute (1999).

TABLE 1.

Salmonella serotypes isolated from imported foods

[FeFe] Hydrogenase Genetic Diversity Provides Insight into Molecular Adaptation in a Saline Microbial Mat Community

Degenerate primers for the [FeFe] hydrogenase (hydA) were developed and used in PCRs to examine hydA in microbial mats that inhabit saltern evaporative ponds in Guerrero Negro (GN), Mexico. A diversity of deduced HydA was discovered that revealed unique variants, which may reflect adaptation to the environmental conditions present in GN.Hydrogen (H₂) is an important intermediate in the decomposition of organic matter in anaerobic environments and is the basis for many syntrophic interactions that occur in the food chain such as those between acetogens and methanogens and/or sulfate reducers. Little is known concerning the potential role of fermentative bacteria and/or H₂ cycling in phototrophic microbial mat systems, such as the 6-cm-thick and extremely diverse microbial mats inhabiting the saline ponds at the Exportadora de Sal SA saltern in Guerrero Negro (GN), Baja California Sur, Mexico. Recent 16S rRNA gene surveys in GN revealed an abundance of bacteria in the upper 2 mm of the mat that, based on their phylogenetic affiliation, are thought to harbor fermentative metabolisms (6, 8, 13, 16, 20, 28). Similarly, previous studies have shown that the flux of H₂ from the surface of the phototrophic mats present in GN is ∼150-fold higher during the night than during the day (9). Together, these results suggest the potential for fermentative metabolisms in H₂ and carbon cycling in the GN saline mat environment.The [FeFe] hydrogenase has a central role in primary and secondary substrate fermentations, catalyzing the oxidation of excess reducing equivalents coupled to the reduction of protons, yielding H₂ (2, 17, 29, 30). The [FeFe] hydrogenase is only known to occur in anaerobic bacteria and a small number of green algae (23, 29, 30), making this a useful biomarker for examining the diversity and distribution of this functional class of organism. Eighty-two putative [FeFe] hydrogenase large-subunit protein sequences (HydA) present in the GenBank database that represent the known diversity of putative HydA were screened for the L1 ([FLI]TSC[C/S]P[GAS]W[VIQH]) and L2 ([IVLF]MPCx[ASRD]K[KQ]xE) (conserved residues are in bold and underlined, and bracketed positions indicate “semiconserved” residues at that position) signature sequence motifs (17, 30). Degenerate PCR primers, corresponding to positions 272 to 279 (AD[M/L]TIMEE) and positions 420 to 427 (TGGVMEAA) in the Clostridium pasteurianum protein sequence, were designed for use in specific gene amplifications.Mat core samples (1 by 6 cm) were collected at 2:00 pm from pond 4 (35°C, pH 8.0, and 80 ppt salt) near pond 5 at the Exportadora de Sal saltworks, GN, on 13 February 2005. Mat cores were sectioned in 1-mm increments on site and immediately flash frozen in liquid nitrogen as previously described (16). Genomic DNA was extracted by bead beating in the presence of phenol-chloroform-isoamyl alcohol and sodium dodecyl sulfate as previously described (7) and was quantified by using the High DNA Mass Ladder (Invitrogen, Carlsbad, CA). Approximately 500-bp fragments of hydA were amplified in triplicate using primers FeFe-272F (5′-GCHGAYMTBACHATWATGGARGA-3′, where H = A, C, T; Y = C, T; M = A, C; B = C, G, T; W = A, T; R = A, G; 432-fold degeneracy) and FeFe-427R (5′-GCNGCYTCCATDACDCCDCCNGT-3′, where N = A, C, T, G; Y = C, T; D = A, G, T; 864-fold degeneracy) and 35 cycles of PCR as previously described (4). An empirically determined annealing temperature of 56.5°C, a MgCl₂ concentration of 1.5 mM, and a primer concentration of 1 μM for each forward and reverse primer were utilized in 50-μl PCR mixtures containing 10 ng of genomic DNA as the template. Equal 40-μl volumes of three replicate PCR products were pooled, purified using the Promega Wizard purification kit (Madison, WI), quantified using the Low Mass DNA Ladder (Invitrogen), cloned using the pGEM Easy Vector System (Promega), and sequenced by using the M13F-M13R primer pair as previously described (5).Deduced amino acid sequences were screened for the presence of the L1 and L2 HydA sequence motifs as described above. ClustalX (version 2.0.8) (15) was employed to align inferred amino acid sequences and to create distance matrices for use in identifying and clustering operational taxonomic units (OTUs) with DOTUR (26). Calculations of Shannon diversity and Chao1 and Ace1 deduced amino acid sequence richness were completed with DOTUR by using the furthest-neighbor algorithm and a precision of 0.01. The phylogenetic position of putative HydA was assessed with MRBAYES (10, 25). Tree topologies were sampled every 500 generations for 2,000,000 generations (burnin = 1,000,000) by using the WAG evolutionary model with fixed amino acid frequencies and gamma-shaped rate variation with a proportion of invariable sites as recommended by ProtTest (1). The Saccharomyces cerevisiae Narf protein, a homolog of HydA, was used as the outgroup. The phylogenetic tree was projected using TreeView (version 1.6.6) (21).An unexpectedly diverse assemblage of putative HydA variants was present in the top 1 mm of the GN microbial mat, corresponding to the photic zone. At a sequence identity threshold (SIT) of 100%, the predicted Shannon diversity index was 3.84 and the mean Chao1 HydA richness was 187 unique OTUs (see Fig. S1a in the supplemental material). When a 99% SIT was applied, the predicted Chao1 richness estimate decreased 56% to 82 unique OTUs and the Shannon diversity index decreased slightly to 3.55 (data not shown). A further decrease in the SIT from 99 to 85% resulted in a slight decrease (23%) in the Chao1 predicted richness and a modest decrease in the Shannon diversity index from 3.55 to 3.28, suggesting that the majority of the putative HydA OTUs present in the top 1 mm of the mat is a result of recent evolution or speciation.The putative HydA variants from GN resolved into 42 OTUs which clustered into one of seven distinct phylogenetic clusters (Fig. ​(Fig.1A),1A), although clusters 5 and 7 could not be adequately resolved. Intriguingly, the mean identity scores determined from sequence alignment matrices for members of each of the seven sequence clusters recovered from GN, when compared to sequences in the GenBank database, were very low (47.5 to 73.2%) (Table ​(Table1),1), suggesting that the putative HydA sequences recovered from GN represent novel sequence space.Open in a separate windowFIG. 1.(A) Phylogenetic tree based on deduced putative HydA amino acid sequences from GN and reference HydA sequences. Nodes with posterior probabilities of <50% were collapsed in this analysis (posterior probabilities of 100 are denoted by asterisks). Bar, one substitution per 10 sites. (B) Partial-length deduced amino acid sequences of environmental and reference deduced HydA sequences illustrating the phylogenetic coherence of novel substitutions and insertions in and upstream of L1 sequence motif. Cluster designations (C1 to C7) correspond to those presented in Table ​Table11.

TABLE 1.

Phylogeny of partial-length putative HydA sequence clusters

Hollow-Fiber Ultrafiltration and PCR Detection of Human-Associated Genetic Markers from Various Types of Surface Water in Florida

Hollow-fiber ultrafiltration (HFUF) and PCR were combined to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF allowed marker detection when membrane filtration did not, demonstrating HFUF''s ability to facilitate detection of diluted targets by PCR in a variety of water types.Microbial source tracking (MST) uses analytical methods to determine the possible source(s) of fecal contamination in environmental waters (reviewed in reference 14). Human fecal pollution (i.e., sewage) is known to contain pathogens that pose serious health risks (6, 8). Identifying sewage impacts can prevent illness, through public warnings and environmental remediation. Detection of sewage has been demonstrated with genetic markers from microorganisms associated with humans (3, 4, 7, 11, 15), each of which has pros and cons (5, 14). A useful addition to MST would be the concentration of samples when the standard protocol (e.g., filtering 500 ml) (5) is not feasible due to turbidity of the water or dilution of the target(s). Hollow-fiber ultrafiltration (HFUF) concentrates particulates from water, with little increase in total assay time (9), while simultaneously retaining parasites, bacteria, and viruses (13). Dead-end HFUF of culturable enterococci in recreational water was successfully demonstrated in Florida (9) and California (10). The objective of this study was to assess the utility of HFUF for concentration of higher-volume samples that would permit detection of diluted MST markers from surface water samples by PCR. Three markers representing a variety of microbial types were targeted: (i) human-associated Bacteroidales (Bacteria), (ii) human polyomaviruses (HPyVs; virus), and (iii) Methanobrevibacter smithii (Archaea).Water samples were collected in Hillsborough Country, FL. Freshwater originated from the unimpacted headwaters of the Hillsborough River (28°5′56.98"N, 82°18′44.05"W) and estuarine water from remediated Ben T. Davis Beach (27°58′5.71"N, 82°34′26.46"W). Sewage-impacted water samples were collected from Sweetwater Creek (27°59′57.27"N, 82°33′37.10"W), which received approximately 500,000 gallons of sewage from a sewer main break. Samples were collected within 5 days and 45 days of the spill. The 5-day sample was collected after the spill was reported, while the 45-day sample was collected to test a more diluted sample of the same water. Raw sewage (influent) for spiked samples was collected on the day of the HFUF experiment from a Hillsborough County wastewater treatment plant that processes 9 million gallons daily.Initial HFUF samples consisted of Dulbecco''s phosphate-buffered saline (D-PBS; pH 7.4) spiked with 5 ml of raw sewage (5 × 10⁻⁴ dilution). Spiked environmental samples included 10 liters of ambient water spiked with 5 or 2.5 ml of raw sewage. Fecal indicator bacterium (FIB) concentrations of the unamended samples were determined by membrane filtration using standard methods for Enterococcus spp., Escherichia coli (16), and fecal coliforms (2) to demonstrate the ambient microbial water quality at the sites. Water samples were first tested for existing MST markers before spiking with sewage. Ten-liter samples of unamended ambient water (Ben T. Davis Beach and Sweetwater Creek) were collected to assess the method''s ability to detect diluted targets following method verification with spiked experiments. Subsequent sewage spikes into unamended samples were performed to ensure that any lack of detection using the HFUF protocol was not due to inhibition. Water samples were collected for processing by the standard protocols from each site in concert with HFUF samples (5). The Sweetwater Creek sample allowed only 300 ml to be filtered instead of the standard 500 ml due to its high turbidity (160 nephelometric turbidity units [NTU]).The design and operation of the recreational dead-end concentrator (Rec DEC) have been previously described (9, 10). A new F80A Hemoflow polysulfone high-flux capillary dialyzer (Fresenius Medical Care North America, Lexington, MA) was used for each sample. Samples were fed into the filters by a peristaltic pump in approximately 6.5 min. All Rec DEC components were cleaned between sample feedings with 10% bleach followed by 10% sodium thiosulfate. Elution of each filter was performed using 250 ml of 4 M urea-50 mM lysine (pH 9.0) that was incubated in the filter fibers for 2 min before collection of the retentate.The pH of each retentate was adjusted to 3.5 to retain HPyVs (11, 12), and then 25 ml was filtered through a 0.45-μm-pore-size nitrocellulose membrane. Each membrane was placed into a Mo Bio PowerSoil microcentrifuge tube (Mo Bio Laboratories, Carlsbad, CA), and extraction of total DNA was performed as previously described (5). Conventional PCR was used to determine the presence or absence of MST markers. Assays targeted a species-specific region of the 16S rRNA gene of human-associated Bacteroidales (3) with slight modifications (5), the conserved T antigen of human BK and JC polyomaviruses (1, 5), or the nifH gene of Methanobrevibacter smithii (5, 15).Each marker was detected in all HFUF spiked samples (Table ​(Table1).1). The procedure was also successful in detecting the markers with the reduced sewage spike volume (2.5 ml) for the Ben T. Davis Beach and Sweetwater Creek samples (Table ​(Table1).1). Detection of smaller amounts of spiked sewage was not attempted in this study; however, previous work determined detection limits of 10⁻³ to 10⁻⁴ dilution for HPyVs and M. smithii (5) and 10⁻⁵ to 10⁻⁶ dilution for Bacteroidales (3, 5).

TABLE 1.

PCR detection of human-associated MST markers from surface water samples following HFUF with Rec DEC