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1.
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
StrainHostDiseaseDistributionFusion cleavagecGI accession no.
APMV1/Newcastle disease virus>250 speciesHigh mortalityWorldwideGRRQKRF45511218
InapparentWorldwideGGRQGRLa11545722
APMV2/Chicken/CA/Yucaipa/1956Turkey, chickens, psittacines, rails, passerinesDecrease in egg production and respiratory diseaseWorldwideDKPASRF169144527
APMV3/Turkey/WI/1968TurkeyMild respiratory disease and moderate egg decreaseWorldwidePRPSGRLa209484147
APMV3/Parakeet/Netherlands/449/1975Psittacines, passerines, flamingosNeurological, enteric, and respiratory diseaseWorldwideARPRGRLa171472314
APMV4/Duck/Hong Kong/D3/1975Duck, geese, chickensNone knownWorldwideVDIQPRF210076708
APMV5/Budgerigar/Japan/Kunitachi/1974Budgerigars, lorikeetsHigh mortality, enteric diseaseJapan, United Kingdom, AustraliaGKRKKRFa290563909
APMV6/Duck/Hong Kong/199/1977Ducks, geese, turkeysMild respiratory disease and increased mortality in turkeysWorldwidePAPEPRLb15081567
APMV7/Dove/TN/4/1975Pigeons, doves, turkeysMild respiratory disease in turkeysUnited States, England, JapanTLPSSRF224979458
APMV8/Goose/DE/1053/1976Ducks, geeseNone knownUnited States, JapanTYPQTRLa226343050
APMV9/Duck/NY/22/1978DucksNone knownWorldwideRIREGRIa217068693
APMV10/Penguin/Falkland Islands/324/2007Rockhopper penguinsNone KnownFalkland IslandsDKPSQRIa300432141
Open in a separate windowaRequires the addition of an exogenous protease.bProtease requirement depends on the isolate examined.cPutative.Six of these serotypes were classified in the latter half of the 1970s, when the most reliable assay available to classify paramyxoviruses was the hemagglutination inhibition (HI) assay (61). However, there are multiple problems associated with the use of serology, including the inability to classify some APMVs by comparing them to the sera of the nine defined APMVs alone (2, 8). In addition, one-way antigenicity and cross-reactivity between different serotypes have been documented for many years (4, 5, 14, 25, 29, 33, 34, 41, 51, 52, 60). The ability of APMVs, like other viruses, to show antigenic drift as it evolves over time (37, 43, 54) and the wide use and availability of precise molecular methods, such as PCR and genome sequencing, demonstrate the need for a more practical classification system.The genetic diversity of APMVs is still largely unexplored, as hundreds of avian species have never been surveyed for the presence of viruses that do not cause significant signs of disease or are not economically important. The emergence of H5N1 highly pathogenic avian influenza (HPAI) virus as the cause of the largest outbreak of a virulent virus in poultry in the past 100 years has spurred the development of surveillance programs to better understand the ecology of avian influenza (AI) viruses in aquatic birds around the globe, and in some instances it has provided opportunities for observing other viruses in wild bird populations (15, 53). In 2007, as part of a seabird health surveillance program in the Falkland Islands (Islas Malvinas), oral and cloacal swabs and serum were collected from rockhopper penguins (Eudyptes chrysocome) and environmental/fecal swab pools were collected from other seabirds.While AI virus has not yet been isolated from penguins in the sub-Antarctic and Antarctic areas, there have been two reports of serum antibodies positive to H7 and H10 from the Adélie species (11, 40). Rare isolations of APMV1, both virulent (45) and of low virulence (8), have been reported from Antarctic penguins. Sera positive for APMV1 and AMPV2 have also been reported (21, 24, 38, 40, 53). Since 1981, paramyxoviruses have been isolated from king penguins (Aptenodytes patagonicus), royal penguins (Eudyptes schlegeli), and Adélie penguins (Pygoscelis adeliae) from Antarctica and little blue penguins (Eudyptula minor) from Australia that cannot be identified as belonging to APMV1 to -9 and have not yet been classified (8, 11, 38-40). The morphology, biological and genomic characteristics, and antigenic relatedness of an APMV recently isolated from multiple penguin colonies on the Falkland Islands are reported here. Evidence that the virus belongs to a new serotype (APMV10) and a demonstration of the advantages of a whole genome system of analysis based on random sequencing followed by comparison of genetic distances are presented. Only after all APMVs are reported and classified will epidemiological information be known as to how the viruses are moving and spreading as the birds travel and interact with other avian species.  相似文献   

2.
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
ClassaNo.Consensus motif sequencesbMaster DNA invertase genecRegulated genesSource or reference(s)
I14ARACGTWCGTBF2765 (mpi)Capsular polysaccharide biosynthesis genes8
II10AGTTC{N5}GAACTBF0667susC/susD paralogsThis study
III3GTTAC{N7}GTAACBF3038, BF4033, BF4283Putative outer membrane protein genes36
IV2TACTTANTAGGTAANAGAABF2766Extracellular polysacharide biosynthesis genes6, 26, 29
V1TCTGCAAAGNCTTTGCAGABF0667susC/susD paralogsThis study
VI1ACTAAGTTCTATCGGBF0667susC/susD paralogsThis study
Open in a separate windowaOur previous classification of the invertible regions identified in B. fragilis strain YCH46 genome (19).bConsensus motif sequences found within IRs are shown. R = A or G, W = A or T, and N = A, G, C, or T.cThe gene identifications in B. fragilis strain YCH46 genome are shown.Our previous studies indicated that an additional inverlon other than the Mpi- and Tsr19-regulated inverlons is present in B. fragilis, based on the finding that at least 10 invertible regions (corresponding to class II regions in B. fragilis strain YCH46) contain a particular consensus motif sequence (AAGTTCN5GAACTT) within their IRs (19) but do not appear to colocalize with a DNA invertase gene. The majority of the class II regions were associated with the selective switching of a particular set of susC/susD family genes. Since the SusC/SusD family of outer membrane proteins play an important role in polysaccharide utilization by Bacteroides (3, 23, 32), the inverlon associated with the phase variation of SusC/SusD family proteins would likely be involved in the survival of this anaerobe in the distal gut.In the present study, we sought to identify the DNA invertase regulating the additional inverlon in B. fragilis. Our results indicated that the Tsr encoded by BF0667 is a master DNA invertase for this inverlon (designated the Tsr0667-inverlon) in B. fragilis.  相似文献   

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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 pathwaysa
Regulator or substrateFunctionReference(s)
Regulators (upstream function)
    LKB1Wnt signaling, Peutz-Jeghers syndrome, insulin signal transduction, pattern formation2, 63, 93
    TAO1MEK3/p38 stress-responsive mitogen-activated protein kinase (MAPK) pathway46
    MARKKNerve growth factor signaling in neurite development and differentiation98
    aPKCCa2+/DAG-independent signal transduction, cell polarity, glucose metabolism14, 37, 40, 45, 59, 75, 95
    nPKC/PKDDAG-dependent, Ca2+-independent signal transduction (GPCR)101
    PAR-3/PAR-6/aPKC(−); regulates Par-1, assembly of microtubules, axon-dendrite specification19
    GSK3β(−); tau phosphorylation, Alzheimer''s dementia, energy metabolism, body patterning54, 97
    Pim-1 oncogene(−); G2/M checkpoint, effector of cytokine signaling and Jak/STAT(3/5)5
    CaMKI(−); Ca2+-dependent signal transduction, neuronal differentiation99
Substrates (downstream function)
    Cdc25CRegulation of mitotic entry by activation of the cdc2-cyclin B complex25, 72, 78, 103
    Class II HDACControl of gene expression and master regulator of subcellular trafficking28, 50
    CRTC2/TORC2Gluconeogenesis regulator via LKB1/AMPK/TORC2 signaling, PPARγ1a coactivator49
    Dlg/PSD-95Synaptogenesis and neuromuscular junction, tumor suppressor (102)104
    DisheveledWnt signaling, translocation of Dsh from cytoplasmic vesicles to cortex73, 94
    KSR1Regulation of the Ras-MAPK pathway68, 69
    MAP2/4/TAUDynamic instability (67, 83) of microtubules, Alzheimer''s dementia (30)11, 31-33, 47, 70, 96
    Mib/NotchMind bomb (Mib degradation and repression of Notch signaling results in neurogenesis)57, 74, 81
    Par3/OSKAR/LglCytoplasmic protein segregation, cell polarity, and asymmetric cell division7, 10
    Pkp2Desmosome assembly and organization; nuclear shuttling68, 69
    PTPH1Linkage between Ser/Thr and Tyr phosphorylation-dependent signaling103
    Rab11-FIPRegulation of endocytosis (23), trafficking of E-cadherin (64)34
Open in a separate windowaLKB1 also is known as Par-4; MARKK also is known as Ste20-like; (−), inhibitory/negative regulation has been shown; GPCR, G protein-coupled receptors. MARKK is highly homologous to TAO-1 (thousand-and-one amino acid kinase) (46).The mammalian Par-1 family contains four members (Table (Table2).2). Physiological functions of the Par-1b kinase have been studied using targeted gene knockout approaches in mice (9, 44). Two independently derived mouse lines null for Par-1b have implicated this protein kinase in diverse physiological processes, including fertility (9), immune system homeostasis (44), learning and memory (86), the positioning of nuclei in pancreatic beta cells (35, 38), and growth and metabolism (43).

TABLE 2.

Terminology and localization of mammalian Par-1 family members
SynonymsaSubcellular localization
Par-1a, MARK3, C-TAK1, p78/KP78, 1600015G02Rik, A430080F22Rik, Emk2, ETK-1, KIAA4230, mKIAA1860, mKIAA4230, M80359Basolateralb/apicalc
Par-1b, EMK, MARK2, AU024026, mKIAA4207Basolateral
Par1c, MARK1Basolateral
Par1d, MARK4, MARKL1Not asymmetricd
Open in a separate windowaPar should not to be confused with protease-activated receptor 1 (PAR1 [29]); C-TAK1, Cdc twenty-five C-associated kinase 1; MARK, microtubule affinity regulating kinase; MARKL, MAP/microtubule affinity-regulating kinase-like 1.bBasolateral to a lesser degree than Par-1b (37).cHuman KP78 is asymmetrically localized to the apical surface of epithelial cells (76).dVariant that does not show asymmetric localization in epithelial cells when overexpressed (95).Beyond Par-1b, most information regarding the cell biological functions of the Par-1 kinases comes from studies of Par-1a. Specifically, Par-1a has been implicated in pancreatic (76) and hepatocarcinogenesis (51), as well as colorectal tumors (77), hippocampal function (100), CagA (Helicobacter pylori)-associated epithelial cell polarity disruption (82), and Peutz-Jeghers syndrome (48), although the latter association has been excluded recently (27). As a first step toward determining unique and redundant functions of Par-1 family members, mice disrupted for a second member of the family (Par-1a/MARK3/C-TAK1) were generated. We report that Par-1a−/− mice are viable and develop normally, and adult mice are hypermetabolic, have decreased white and brown adipose tissue mass, and unaltered glucose/insulin handling. However, when challenged by a high-fat diet (HFD), Par-1a−/− mice exhibit resistance to hepatic steatosis, resistance to glucose intolerance, and the delayed onset of obesity relative to that of control littermates. Strikingly, overnight starvation results in a complete depletion of glycogen and lipid stores along with an increase in autophagic vacuoles in the liver of Par-1a−/− but not Par-1b−/− mice. Correspondingly, Par-1a−/− mice develop hypoketotic hypoglycemia. These findings reveal unique metabolic functions of two Par-1 family members.  相似文献   

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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
Organism abbreviation used in textSpeciesStrainGeographic originReference(s)
A9Myxococcus xanthusA9Tübingen, Germany78
A23Myxococcus xanthusA23Tübingen, Germany78
A30Myxococcus xanthusA30Tübingen, Germany78
A41Myxococcus xanthusA41Tübingen, Germany78
A46Myxococcus xanthusA46Tübingen, Germany78
A47Myxococcus xanthusA47Tübingen, Germany78
A75Myxococcus xanthusA75Tübingen, Germany78
A85Myxococcus xanthusA85Tübingen, Germany78
TVMyxococcus xanthusTvärminneTvärminne, Finland79
PAKMyxococcus xanthusPaklenicaPaklenica, Croatia79
MADMyxococcus xanthusMadeira 1Madeira, Portugal79
WARMyxococcus xanthusWarwick 1Warwick, UK79
TORMyxococcus xanthusToronto 1Toronto, Ontario, Canada79
SUL2Myxococcus xanthusSulawesi 2Sulawesi, Indonesia79
KALMyxococcus xanthusKalalauKalalau, HI79
DAVMyxococcus xanthusDavis 1ADavis, CA79
GJV1Myxococcus xanthusGJV 1Unknown35, 72
MXFL1Myxococcus flavescensMx fl1Unknown65
MXV2Myxococcus virescensMx v2Unknown65
CCM8Myxococcus macrosporusCc m8Unknown65
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Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.The genus Mycobacterium has a unique feature in the cell envelope that contains a multilayered structure consisting of peptidoglycan, mycolyl-arabinogalactan complex, and surface glycolipids (8, 12). It is known that these components play a role in protection from environmental stresses, such as antimicrobial agents and host immune responses (8, 12). Some of them are recognized as pathogenic factors related to mycobacterial diseases, such as tuberculosis and leprosy (8, 12). In case of nontuberculous mycobacteria that are widely distributed in the natural environment as opportunistic pathogens, glycopeptidolipids (GPLs) are abundantly present on the cell envelope as surface glycolipids (34). GPLs have a core structure in which a fatty acyl-tetrapeptide is glycosylated with 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha) (2, 5, 13). This structure is common to all types of GPLs, and GPLs with this structure that have not undergone further glycosylation are termed non-serovar-specific GPLs (nsGPLs) (2, 5, 13). Structural diversity generated by further glycosylations, such as rhamnosylation, fucosylation, and glucosylation, is observed for the oligosaccharide portion linked to the 6-d-Tal residue of nsGPLs from Mycobacterium avium complex (MAC), a member of the nontuberculous mycobacteria consisting of two species, M. avium and M. intracellulare (2, 5, 34). Consequently, these nsGPLs with varied oligosaccharides lead to the formation of the serovar-specific GPLs (ssGPLs) that define around 30 types of MAC serovars (10).The properties of MAC serovars are known to be notably different from each other and also to be closely associated with the pathogenicity of MAC (3, 6, 18, 30, 31, 32). Various epidemiological studies indicate that serovar 4 is the most prevalent type and is also one of the serovars frequently isolated from AIDS patients (1, 20, 33, 36). Additionally, pulmonary MAC disease caused by serovar 4 is shown to exhibit a poorer prognosis than that caused by other serovars (23). With respect to host immune responses to MAC infection, serovar 4-specific GPL is reported to have characteristic features that are in contrast to those of other ssGPLs (21, 30). Structurally, serovar 4-specific GPL contains a unique oligosaccharide in which the oligosaccharide of serovar 2-specific GPL is further glycosylated with 4-O-methyl-rhamnose (4-O-Me-Rha) residue through a 1→4 linkage (Table (Table1)1) (24). Therefore, it is thought that the presence of 4-O-Me-Rha and its linkage position are important in exhibiting the specificity of biological activities. The biosynthesis of the oligosaccharide portion in several ssGPLs is currently being clarified (15, 16, 17, 25, 26), while that of serovar 4-specific GPL is still unresolved. In this study, we have focused on the genomic region predicted to be associated with GPL biosynthesis in the serovar 4 strain and explored the key genes responsible for the formation of 4-O-Me-Rha that might determine the specific properties of MAC serovar 4.

TABLE 1.

Oligosaccharide structures of serovar 2- and 4-specific GPLs
SerovarOligosaccharideReference
22,3-di-O-Me-α-l-Fuc-(1→3)-α-l-Rha-(1→2)-l-6-d-Tal9
44-O-Me-α-l-Rha-(1→4)-2-O-Me-α-l-Fuc-(1→3)-α-l-Rha-(1→2)-l-6-d-Tal24
Open in a separate window  相似文献   

14.
15.
16.
17.
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
AAVAmino acid at positiona:
Glycan targetbReference
129418531532584598642
AAV1LEEDFANS70
AAV1-E/KLEKDFANHS+ (and S)c70
AAV6FDKDLVHHS and S70
AAV6.1FDEDLVHHS (and S)c40, 70
AAV6.2LDKDLVHHS (and S)c40, 70
AAV6R2LDEDLVHHS (and S)c40
HAE1LEEDLVN(HS and S)d39
HAE2LDKDLVN(HS and S)d39
shH10FDKNLVNHS (and S-inde)33
Open in a separate windowaMutant residues in boldface have an AAV6 parental original; those underlined have an AAV1 parental origin.bS, sialic acid; HS, heparan sulfate; HS+, HS positive.cThe sialic acid binding phenotypes of these mutants were not discussed in the respective publications but are assumed to be still present.dThe glycan targets for these mutants were not discussed in this publication; thus, the phenotypes indicated are assumed.eThis mutant is sialic acid independent (S-ind) for cellular transduction.The structures of AAV1 to AAV5 and AAV8 have been determined by X-ray crystallography and/or cryo-electron microscopy and image reconstruction (cryo-EM) (23, 36, 47, 52, 66, 73; unpublished data), and preliminary characterization of crystals has also been reported for AAV1, AAV5, AAV7, and AAV9 (15, 45, 46, 55). The capsid VP structures contain a conserved eight-stranded (βB to βI) β-barrel core and large loop regions between the strands that form the capsid surface. The capsid surface is characterized by depressions at the icosahedral 2-fold axes of symmetry, finger-like projections surrounding the 3-fold axes, and canyon-like depressions surrounding the 5-fold axes. A total of nine variable regions (VRs; VRI to VRIX) were defined when the two most disparate structures, AAV2 and AAV4, were compared (23). The VRs contain amino acids that contribute to slight differences in surface topologies and distinct functional phenotypes, such as in receptor binding, transduction efficiency, and antigenic reactivity (10, 23, 37, 47).The structure of virus-like particles (VLPs) of AAV6, produced in a baculovirus/Sf9 insect cell expression system, has been determined by two highly complementary approaches, cryo-EM and X-ray crystallography. The AAV6 VP structure contains the general features already described for the AAVs and has conformational differences in the VRs compared to the VRs of other AAVs. The 9.7-Å-resolution cryoreconstructed structure enabled the localization of the C-α positions of five of the six amino acids that differ between highly homologous AAV6 and AAV1 but did not provide information on the positions of the side chains or their orientations. The X-ray crystal structure determined to 3.0-Å resolution enabled us to precisely map the atomic positions of these five residues at or close to the icosahedral 3-fold axes of the capsid. Reported mutagenesis and biochemical studies had functionally annotated the six residues differing between AAV1 and AAV6 with respect to their roles in receptor attachment and differential cellular transduction. Their disposition identifies the 3-fold capsid region as playing essential roles in AAV infection.  相似文献   

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

TABLE 1.

Nitrogen mass balance
Time (h)% of total initial nitrogen by mass recovered ina:
Total recovery (%)
1MNG2MNG1,2DNG1,3DNGNGProteinNitriteNitrate
0NDbND0.9 ± 0.70.8 ± 0.682 ± 5.20.8 ± 0.214 ± 0.70.8 ± 0.3100 ± 5.3
460.1 ± 0.00.8 ± 0.27.9 ± 0.4ND35 ± 3.62.0 ± 0.549 ± 1.11.7 ± 0.096 ± 4.2
720.1 ± 0.00.9 ± 0.24.3 ± 4.2ND5.0 ± 0.43.3 ± 0.281 ± 4.23.9 ± 1.998 ± 6.8
94ND0.6 ± 0.4NDND0.6 ± 0.43.2 ± 0.095 ± 102.6 ± 1.6102 ± 10
Open in a separate windowaData represent averages of four replicates ± standard deviations.bND, not detected.

TABLE 2.

Carbon mass balance
Time (h)% of total initial carbon by mass recovered in:
Total recovery (%)
1MNGa2MNGa1,2DNGa1,3DNGaNGaProteinaCO2b
0NDcND1.6 ± 1.21.9 ± 0.492 ± 5.84.4 ± 0.9100 ± 8.4
460.5 ± 0.22.6 ± 0.613 ± 0.7ND39 ± 3.913 ± 3.028 ± 5.796 ± 14.1
720.4 ± 0.02.9 ± 0.77.3 ± 7.0ND5.6 ± 0.422 ± 1.259 ± 8.397 ± 17.6
94ND2.8 ± 0.3NDND0.8 ± 0.518 ± 0.371 ± 4.593 ± 5.6
Open in a separate windowaData represent averages of four replicates ± standard deviations.bData represent averages of duplicates ± standard deviations.cND, not detected.In a separate experiment cells grown on NG were added to minimal media containing 1,3DNG, 1,2DNG, 1MNG, or 2MNG and degradation over time was measured. 1,2DNG, 1,3DNG, and 1MNG were degraded at rates of 6.5, 3.8, and 8 μmol substrate/mg protein/hour. No degradation of 2MNG was detected (after 250 h), which indicates that 2MNG is not an intermediate in a productive degradation pathway. Because 1,3DNG was not observed at any point during the degradation of NG and its degradation rate is approximately one-half the degradation rate of 1,2DNG, it also seems not to be part of the main NG degradation pathway used by Arthrobacter sp. strain JBH1. The above observations indicate that the degradation pathway involves a sequential denitration of NG to 1,2DNG, 1MNG, and then glycerol, which serves as the source of carbon and energy (Fig. (Fig.2).2). The productive degradation pathway differs from that observed by previous authors using both mixed (1, 3, 6) and pure cultures (4, 9, 11, 20), in which both 1,3- and 1,2DNG were intermediates during NG transformation. Additionally, in previous studies both MNG isomers were produced regardless of the ratio of 1,2DNG to 1,3DNG (3, 4, 6, 9, 20). Our results indicate that the enzymes involved in denitration of NG in strain JBH1 are highly specific and catalyze sequential denitrations that do not involve 1,3DNG or 2MNG. Determination of how the specificity avoids misrouting of intermediates will require purification and characterization of the enzyme(s) involved.Open in a separate windowFIG. 2.Proposed NG degradation pathway.Mass balances of carbon and nitrogen were used to determine the following stoichiometric equation that describes NG mineralization by Arthrobacter sp. strain JBH1: 0.26C3H5(ONO2)3 + 0.33O2 → 0.03C5H7O2N + 0.63CO2 + 0.75NO2 + 0.75H+ + 0.17H2O. The result indicates that most of the NG molecule is being used for energy. The biomass yield is relatively low (0.057 mg protein/mg NG), with an fs (fraction of reducing equivalents of electron donor used for protein synthesis) of 0.36 (10), which is low compared to the aerobic degradation of other compounds by pure cultures, for which fs ranges between 0.4 and 0.6 (10, 14). The results are consistent with the requirement for relatively large amounts of energy during the initiation of the degradation mechanism (each denitration probably requires 1 mole of NADH or NADPH [21]).Although NG degradation rates were optimal at pH 7.2, they were still substantial at values as low as 5.1. The results suggest that NG degradation is possible even at low pH values typical of the subsurface at sites where explosives were formerly manufactured or sites where nitrite production lowers the pH.NG concentrations above 0.5 mM are inhibitory, but degradation was still observed at 1.2 mM (data not shown). The finding that NG can be inhibitory to bacteria at concentrations that are well below the solubility of the compound is consistent with those of Accashian et al. (1) for a mixed culture.The ability of Arthrobacter sp. strain JBH1 to grow on NG as the carbon and nitrogen source provides the basis for a shift in potential strategies for natural attenuation and bioremediation of NG at contaminated sites. The apparent specificity of the denitration steps raises interesting questions about the evolution of the pathway.  相似文献   

19.
Bacterial biofilms are responsible for the majority of all microbial infections and have profound impact on industrial and geochemical processes. While many studies documented phenotypic differentiation and gene regulation of biofilms, the importance of their structural and mechanical properties is poorly understood. Here we investigate how changes in lipopolysaccharide (LPS) core capping in Pseudomonas aeruginosa affect biofilm structure through modification of adhesive, cohesive, and viscoelastic properties at an early stage of biofilm development. Microbead force spectroscopy and atomic force microscopy were used to characterize P. aeruginosa biofilm interactions with either glass substrata or bacterial lawns. Using isogenic migA, wapR, and rmlC mutants with defined LPS characteristics, we observed significant changes in cell mechanical properties among these strains compared to wild-type strain PAO1. Specifically, truncation of core oligosaccharides enhanced both adhesive and cohesive forces by up to 10-fold, whereas changes in instantaneous elasticity were correlated with the presence of O antigen. Using confocal laser scanning microscopy to quantify biofilm structural changes with respect to differences in LPS core capping, we observed that textural parameters varied with adhesion or the inverse of cohesion, while areal and volumetric parameters were linked to adhesion, cohesion, or the balance between them. In conclusion, this report demonstrated for the first time that changes in LPS expression resulted in quantifiable cellular mechanical changes that were correlated with structural changes in bacterial biofilms. Thus, the interplay between architectural and functional properties may be an important contributor to bacterial community survival.Biofilms are sessile microbial communities growing on a surface or at an interface, often enmeshed in polymeric substances. Being the predominant mode of microbial growth in nature, bacterial biofilms are particularly problematic in the context of human health, accounting for up to 80% of all bacterial infections. In industrial processes, bacterial biofilms cause corrosion and biofouling, resulting in considerable loss of productivity. In the natural environment, biofilms play a role in modulating worldwide geochemical cycles. Given the impact of biofilms in these diverse areas, the need for developing effective strategies to control them is of paramount importance. Since bacterial cell surface structures are convenient targets for control agents, their roles in influencing biofilm function and architecture warrant in-depth investigations. To date, most studies of biofilms have focused on genetic regulation, phenotypic differentiation and their contribution to antibiotic resistance. In contrast, the mechanical and structural properties that link the genotypes to phenotypes of bacterial biofilms are not well understood and rarely studied in a quantitative and correlated manner.Pseudomonas aeruginosa is a gram-negative opportunistic pathogen implicated in serious infections in patients with cystic fibrosis and immunocompromised patients. This bacterium has a relatively large genome (6.3 Mb) consistent with its propensity to utilize versatile metabolic pathways, thereby developing antibiotic resistance and producing an arsenal of virulence factors, including lipopolysaccharide (LPS) present on the cell surface. LPS is localized in the outer leaflet of the outer membrane of all gram-negative bacteria, forming the first point of contact between the bacterial cell and any surface that it colonizes or therapeutic agents. The LPS of P. aeruginosa consists of three regions: lipid A, core oligosaccharide (core OS), and O antigen. The O antigen is synthesized as two distinct forms with overlapping pathways: the shorter A-band homopolymer is the so-called “common polysaccharide antigen” among this species and consists of repeating d-rhamnose (d-Rha) subunits, while the longer B-band heteropolymer is composed of repeating tri- to pentasaccharide subunits that vary among the 20 serotypes of P. aeruginosa (42). The core OS is conceptually divided into the highly conserved inner core and the more variable outer core. Depending on the linkage of l-rhamnose (l-Rha) with two distinct d-glucose (d-Glc) residues, two main glycoforms of the core OS exist (see Fig. Fig.1A).1A). In the “capped” glycoform, l-Rha is α-1,3 linked to a d-Glc and acts as the acceptor molecule for O antigen, resulting in the production of smooth LPS. In the “uncapped” glycoform, l-Rha is α-1,6 linked to a different d-Glc and is not substituted with O antigen, resulting in the production of rough LPS (39). In addition, the presence or absence of the α-1,6 linked l-Rha substituted with a terminal d-Glc gives rise to the so-called intact or truncated outer core, respectively. The functional significance of this terminal glucose is unclear at present, although a role in host cell binding has been proposed (57).Open in a separate windowFIG. 1.Comparison of LPSs from Pseudomonas aeruginosa wild-type strain PAO1 and mutant strains with LPS core variants. (A) Schematic diagram illustrating the chemical structures of smooth LPS and rough LPS. The gray and black arrows point to the position of outer core truncation and position of O-antigen capping, respectively. (B) Silver-stained SDS-polyacrylamide gels illustrating LPS profiles of planktonic and biofilm cells. The two gray arrows and the black arrows point to the position of O antigen and position of core-plus-one entities, respectively. Planktonic cells (lanes 1 to 4) and biofilm cells (lanes 5 to 8) of strain PAO1 (P), migA mutant (M), wapR mutant (W), and rmlC mutant (R) are shown.Mechanical processes that are important in the biofilm life cycle include bacterial adhesion, cohesion, and viscoelasticity. Bacterial adhesion is a prerequisite for surface colonization and the most important functional determinant in the early stages of biofilm development. Accurate measurement of adhesion is therefore essential for monitoring the tendency of bacteria to attach to surfaces and to switch from a planktonic lifestyle to a biofilm lifestyle. Data accumulated in previous studies suggest that LPS is involved in bacterial cell adhesion to both abiotic (2, 8, 20, 32, 35, 54, 56) and biotic (17, 38, 49, 56, 57) surfaces. Moreover, environmental factors, such as growth temperature, pH, ionic strength, nutrient availability, and oxygen levels, may influence cell adhesion via modification of LPS expression and conformation (16, 36, 46, 47, 53). The effect of LPS on bacterial adhesion to various types of surfaces apparently involves distinct and complex mechanisms that remain to be elucidated.Bacterial cohesion, herein defined as cell-to-cell adherence, is crucial to the formation of microbial flocs and the growth and detachment of established bacterial biofilms. Quantification of cohesion is important for understanding biofilm biology, and such data are crucial for modeling and forecasting biofilm development so that better control strategies can be developed (55). Previous studies of biofilm cohesiveness have characterized it as highly stratified (3, 4, 18, 43), influenced by ionic strength (14, 34), proportional to shear rate (37), and often variable over 3 orders of magnitude (40, 50). Although an earlier study by Spiers and Rainey (48) provided semiquantitative measurements of the role of LPS on bacterial cohesion within a biofilm, a truly quantitative account of the effect of LPS on biofilm cohesion has not been demonstrated.Bacterial viscoelasticity refers to the combined liquid-like and solid-like characteristics in the behavior of polymeric systems such that when deformed under stress, their strain can increase over time (i.e., creep) and their original shape may be only partially restored upon stress relief (19). Although earlier reports suggested that LPS modulates bacterial cell compressibility and helps prevent catastrophic structural failure due to mechanical stress (1, 52), no direct physical evidence of its involvement in these processes has yet been presented. Therefore, monitoring biofilm viscoelasticity is crucial for demonstrating how well biofilms resist stresses, due to, for instance, fluid shear and antimicrobial peptides (5, 6). To date, quantitative data on how LPS affects viscoelastic properties of biofilms are lacking, and existing studies have merely focused on elasticity measurements (7, 52). Recently, our group has developed an atomic force microscopy (AFM)-based technique called microbead force spectroscopy (MBFS) to measure the adhesive forces and viscoelastic properties of cells within bacterial biofilms (28). In this study, we expand the application of this MBFS method to measure cohesive forces between cells at an early stage of biofilm development.Biofilm structure refers to the distribution of biomass or carbonaceous materials associated with cells (including all viable and nonviable cells and their extracellular polymeric substances) within the space occupied by a biofilm. It is known to be very heterogeneous and highly stratified, typically composed of a cohesive basal layer and a relatively fragile top layer (15, 18, 43). Using confocal laser scanning microscopy (CLSM), biofilm structure can be quantitatively described in terms of textural and volumetric parameters (11, 29). Textural parameters characterize the pattern of cell clusters and interstitial voids in a biofilm, whereas volumetric parameters describe the morphological characteristics of bacterial biofilms in three dimensions (3-D) (11). Biomass distribution is affected by the surrounding environment and may reflect fundamental processes occurring within biofilms, such as nutrient transport, accumulation rate, microbial physiology, and mechanical behavior (29). Therefore, quantifying biofilm structure by CLSM will allow us to understand the underlying processes and the relationship between biofilm architecture and behavior (15).To examine the effects of differential LPS core capping on the mechanical properties of early biofilms (here defined as confluent bacterial lawns that have just begun to develop into full-fledged biofilms) and the structural properties of mature biofilms, we compare P. aeruginosa wild-type strain PAO1 with those of its isogenic migA, wapR, and rmlC mutant strains with defects in the respective genes affecting LPS core biosynthesis (22, 31, 39, 41). The migA gene (PA0705) encodes the putative α-1,6-rhamnosyltransferase necessary for the attachment of the terminal d-Glc to the outer core (39). The wapR gene (PA5000) encodes the putative α-1,3-rhamnosyltransferase crucial to the capping of the core with O antigen (39). The rmlC gene (PA5164) encodes a dTDP-4-dehydrorhamnose 3,5-epimerase essential in the biosynthesis of TDP-l-Rha, which is the precursor for the l-Rha in the LPS core (41). Defects in migA, wapR, and rmlC mutants result in the expression of different LPS phenotypes, including a truncated outer core and/or a lack of capping by O antigen (Table (Table1).1). In this study, we test the hypothesis that LPS contributes to biofilm function and architecture through modulation of cellular mechanics and microcolony structures, thereby contributing to bacterial community survival. By correlating quantitative mechanical changes in early P. aeruginosa biofilms and structural changes in mature biofilms due to differences in LPS chemistry, we aim to elucidate how the properties of these important bacterial cell surface molecules can alter the physical nature of biofilms.

TABLE 1.

Pseudomonas aeruginosa strains used in this study
StrainCharacteristic(s)LPS phenotypebReference
PAO1P. aeruginosa wild-type strain; IATSa serotype O5A+B+; capped, intact outer coreHancock and Carey (22)
migA mutantPAO1-derived migA::Gmr knockout mutantA+B+; capped, truncated outer corePoon et al. (39)
wapR mutantPAO1-derived wapR::Gmr knockout mutantAB; uncapped, intact outer corePoon et al. (39)
rmlC mutantPAO1-derived rmlC::Gmr knockout mutantAB; uncapped, truncated outer coreLindhout et al. (31)
Open in a separate windowaIATS, International Antigenic Typing Scheme.bAB, devoid of A-band and B-band LPS; A+B+, presence of A-band and B-band LPS.  相似文献   

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