Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Coculture Fermentations of Bifidobacterium Species and Bacteroides thetaiotaomicron Reveal a Mechanistic Insight into the Prebiotic Effect of Inulin-Type Fructans

Four bifidobacteria, each representing a cluster of strains with specific inulin-type-fructan degradation capacities, were grown in coculture fermentations with Bacteroides thetaiotaomicron LMG 11262, a strain able to metabolize both oligofructose and inulin. In a medium for colon bacteria with inulin as the sole added energy source, the ability of the bifidobacteria to compete for this substrate reflected phenotypical variation. Bifidobacterium breve Yakult, a strain that was not able to degrade oligofructose or inulin, was outcompeted by B. thetaiotaomicron LMG 11262. Bifidobacterium adolescentis LMG 10734, a strain that could degrade oligofructose (displaying a preferential breakdown mechanism) but that did not grow on inulin, managed to become competitive when oligofructose and short fractions of inulin started to accumulate in the fermentation medium. Bifidobacterium angulatum LMG 11039^T, a strain that was previously shown to degrade all oligofructose fractions simultaneously and to be able to partially break down inulin, was competitive from the beginning of the fermentation, consuming short fractions of inulin from the moment they appeared. Bifidobacterium longum LMG 11047, representing a cluster of bifidobacteria that shared both high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin, was the dominating strain in a coculture with B. thetaiotaomicron LMG 11262. These observations indicate that distinct subgroups within the large-intestinal Bifidobacterium population will be stimulated by different groups of prebiotic inulin-type fructans, a variation that could be reflected in differences concerning their health-promoting effects.The vast complexity of the human colon microbiota, the key element of the large-intestinal ecosystem, has inspired researchers to describe it as a postnatally acquired microbial organ located inside a host organ (1, 46). The microbial colon community is estimated to be composed of up to 100 trillion microorganisms, a number exceeding 10 times the total number of somatic and germ cells of a human adult (18, 38). The human microbiome is thought to contain more than 100 times the total number of human genes (1, 18). It not only broadens the digestive abilities of the host (18, 22, 40) but also influences body processes far beyond digestion (7, 33). In spite of its fundamental impact on human health and disease, the human gastrointestinal ecosystem remains largely unexplored (7, 8).Despite the fact that the present knowledge of the composition of the human large-intestinal microbiota is partial, fragmented, and undetailed, the consistency of some observations allows them to be generalized as facts (8, 28, 47). Notwithstanding the huge diversity at the strain level, up to 87% of the human colon inhabitants belong to only two bacterial phyla, the Bacteroidetes and the Firmicutes (1, 8, 14). Within the group of large-intestinal Bacteroidetes, large variations between individuals have been reported (8). However, Bacteroides spp. generally seem to account for up to 20% of the human colon microbiota (26, 32). Moreover, the presence of Bacteroides thetaiotomicron appears to be universal (8, 21). This species, which has been isolated only from human and rodent intestines or feces up to now, has gained importance as a perfect example of a flexible, niche-adapted, human symbiont with a wide carbohydrate consumption range (3, 4, 40).Although B. thetaiotaomicron is considered a human symbiont contributing to the stability of the colon ecosystem, the Bacteroides genus also harbors some notorious pathogens that are linked with severe extraintestinal infections and that have been mentioned as causal agents of acute diarrhea (30, 35). Moreover, besides their enormous saccharolytic potential, Bacteroides spp. are also capable of proteolytic fermentation (22). These considerations make them unsuited as target organisms for stimulation by prebiotics such as inulin-type fructans (23, 31).Most in vivo studies regarding the effect of the addition of inulin or oligofructose to the diet on the composition of the human colon microbiota reveal that Bacteroides spp. are neither stimulated nor repressed through administration of these prebiotics (34). However, at least some Bacteroides spp. are able to degrade inulin-type fructans, including B. thetaiotaomicron (13, 44). Since this species accounts for up to 6% of the colon microbiota (8), it is at least surprising that its numbers are hardly influenced by an increased availability of these prebiotics as substrates for large-intestinal fermentation. A possible explanation for these contradicting observations is to be found in the mechanism of inulin degradation, which in the case of Bacteroides is presumed to be periplasmic or even extracellular (37, 44). Leakage of free fructose toward the extracellular environment appears to be inherent in such breakdown mechanisms (10, 25, 44). Hence, extracellular fructan degraders inevitably provide opportunistic competitors, which are not able to degrade inulin-type fructans themselves, with a valuable source of energy (2, 10, 19). In contrast, a cell-associated or intracellular degradation mechanism is thought to be widespread among Bifidobacterium spp., which are still considered the main target organisms for prebiotic stimulation by inulin-type fructans (15, 16, 39, 44). This mechanism is often reflected in a clearly preferential breakdown of different-chain-length fractions of oligofructose, which approaches degradation of the long fractions only when short ones are depleted (10, 42, 44). The main disadvantage of such a cell-associated or intracellular degradation strategy seems to be the bifidobacterial incapacity to grow on long-chain-length fractions of inulin (36). Reports of the latter are indeed scarce: kinetic pure culture studies report an upper chain length limit for inulin degradation by Bifidobacterium spp., a disadvantage that will presumably not affect extracellular fructan degraders, such as Bacteroides spp. (9). Although the prebiotic effect of inulin-type fructans on the colon Bifidobacterium population is well documented, in vivo stimulation studies usually tend to consider the bifidobacterial community as a whole, ignoring interspecies differences (23). However, since the early days of in vitro prebiotic studies, a large variation in fructan degradation capacities of different Bifidobacterium strains has been reported (17, 36). It is likely that this variety is translated to the in vivo environment, implying that not all bifidobacteria are equally subject to prebiotic stimulation (5, 45). In a recent study, the kinetics of growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium spp., 17 of which were human intestinal isolates, have been statistically analyzed (9). The existence of four phenotypically distinct clusters among the tested strains, probably reflecting niche-specific adaptation, has been revealed. This rather limited variation was hypothesized to influence the susceptibilities of various bifidobacteria toward prebiotic stimulation by inulin-type fructans and their fitness to compete for these substrates in a complex environment, such as the colon ecosystem (44).The present study aimed at mapping the fructan degradation capacity of B. thetaiotaomicron LMG 11262 growing on oligofructose or inulin. In vitro competitiveness trials with bifidobacterial strains belonging to the different phenotypical clusters mentioned above were designed to investigate the abilities of these strains to compete for inulin in a coculture with an inulin-degrading B. thetaiotaomicron strain.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

A Bacillus anthracis S-Layer Homology Protein That Binds Heme and Mediates Heme Delivery to IsdC

The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.Pathogenic bacteria need to acquire iron to survive in mammalian hosts (12). However, the host sequesters most iron in the porphyrin heme, and heme itself is often bound to proteins such as hemoglobin (14, 28, 85). Circulating hemoglobin can serve as a source of heme-iron for replicating bacteria in infected hosts, but the precise mechanisms of heme extraction, transport, and assimilation remain unclear (25, 46, 79, 86). An understanding of how bacterial pathogens import heme will lead to the development of new anti-infectives that inhibit heme uptake, thereby preventing or treating infections caused by these bacteria (47, 68).The mechanisms of transport of biological molecules into a bacterial cell are influenced by the compositional, structural, and topological makeup of the cell envelope. Gram-negative bacteria utilize specific proteins to transport heme through the outer membrane, periplasm, and inner membrane (83, 84). Instead of an outer membrane and periplasm, Gram-positive bacteria contain a thick cell wall (59, 60). Proteins covalently anchored to the cell wall provide a functional link between extracellular heme reservoirs and intracellular iron utilization pathways (46). In addition, several Gram-positive and Gram-negative bacterial genera also contain an outermost structure termed the S (surface)-layer (75). The S-layer is a crystalline array of protein that surrounds the bacterial cell and may serve a multitude of functions, including maintenance of cell architecture and protection from host immune components (6, 7, 18, 19, 56). In bacterial pathogens that manifest an S-layer, the “force field” function of this structure raises questions concerning how small molecules such as heme can be successfully passed from the extracellular milieu to cell wall proteins for delivery into the cell cytoplasm.Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the etiological agent of anthrax disease (30, 33). The life cycle of B. anthracis begins after a phagocytosed spore germinates into a vegetative cell inside a mammalian host (2, 40, 69, 78). Virulence determinants produced by the vegetative cells facilitate bacterial growth, dissemination to major organ systems, and eventually host death (76-78). The release of aerosolized spores into areas with large concentrations of people is a serious public health concern (30).Heme acquisition in B. anthracis is mediated by the action of IsdX1 and IsdX2, two extracellular hemophores that extract heme from host hemoglobin and deliver the iron-porphyrin to cell wall-localized IsdC (21, 45). Both IsdX1 and IsdX2 harbor near iron transporter domains (NEATs), a conserved protein module found in Gram-positive bacteria that mediates heme uptake from hemoglobin and contributes to bacterial pathogenesis upon infection (3, 8, 21, 31, 44, 46, 49, 50, 67, 81, 86). Hypothesizing that B. anthracis may contain additional mechanisms for heme transport, we provide evidence that B. anthracis S-layer protein K (BslK), an S-layer homology (SLH) and NEAT protein (32, 43), is surface localized and binds and transfers heme to IsdC in a rapid, contact-dependent manner. These results suggest that the Isd system is not a self-contained conduit for heme trafficking and imply that there is functional cross talk between differentially localized NEAT proteins to promote heme uptake during infection.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.Cysteine thiols in proteins fulfill an important and diverse set of cellular functions. In particular, they participate in enzymatic catalysis; in metal coordination, such as in the generation of Fe-S-clusters; and in determining the spatial structure of proteins via disulfide bond formation (3, 22, 23, 38). Cysteines are strong nucleophiles amenable to posttranslational modifications by reactive oxygen species (ROS) and reactive nitrogen species, leading to disulfides; to sulfenic, sulfinic, or sulfonic acids; mixed disulfides with low-molecular-weight (LMW) thiols (S thiolations); and S nitrosylations (7, 16, 17, 27).The redox status of the cytoplasm is under physiological conditions in a reduced state. Thus, most cysteines are present as free thiols (6). Because aerobic organisms have to cope with oxidative stress caused by ROS, such as superoxide anions, hydrogen peroxide, or hydroxyl radicals, they need to employ effective mechanisms that maintain the reduced state. In gram-negative bacteria, the thiol-disulfide balance is accomplished by the glutathione (GSH) system, a thiol-based redox buffer. The GSH system consists of glutaredoxin (Grx), GSH (γ-glutamylcysteinyl glycine), GSH reductase, and GSH peroxidase (34). Reduction of disulfides occurs via sequential electron transfer from glutaredoxin and reduced GSH; oxidized GSH (GSSG) is reduced by the NADPH-dependent GSH reductase. GSH peroxidase enables the direct detoxification of ROS by GSH oxidation.However, many gram-positive bacteria lack genes for GSH biosynthesis. Actinomycetes instead use a thiol redox buffer based on mycothiol (50). Bacillus subtilis, Staphylococcus aureus, and other gram-positive bacteria rely on different thiol redox buffers based on cysteine, the novel 398-Da bacillithiol (BSH), or coenzyme A (CoA) (15, 52). To maintain the reduced state of the cytoplasm, most bacteria use enzymatic systems for disulfide bond reduction such as the thioredoxin (Trx) system, which is highly conserved in gram-negative bacteria (3, 10). The Trx system consists of thioredoxin (TrxA) and the NADPH-dependent thioredoxin reductase (TrxB).Any imbalance in the cellular redox state caused by ROS elicits expression of a repertoire of different proteins, commonly under the control of a redox-sensing regulator: for example, OxyR in Escherichia coli and PerR, OhrR, SarZ, and Spx in B. subtilis and S. aureus, respectively (11, 12, 41, 55, 58, 64-66). The subsequently induced proteins detoxify ROS and restore and protect the normal physiological redox state in the cell.Besides ROS and reactive nitrogen species, so-called “reactive electrophilic species” (RES) affect the thiol redox balance. RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2, 43, 45, 46, 53, 66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine. Exposure of cells to diamide induces the oxidative as well as the electrophile stress response in B. subtilis (43, 45, 53). The toxicity of diamide is based on disulfide bond formation (40), which was recently visualized in B. subtilis and S. aureus by the fluorescence alkylation of oxidized thiols (FALKO) assay (32, 64). It was thought that the formation of nonnative inter- and intramolecular disulfide bonds results in damage of proteins.However, more recent findings demonstrate that diamide stress leads also to S thiolations: formation of disulfide bonds between proteins and LMW thiols (8, 13, 33). S thiolations prevent protein thiols from irreversible oxidation to sulfinic and sulfonic acids, but also affect enzyme activity (35, 47) and signal transduction (39, 42). In B. subtilis, we have identified a few cytoplasmic proteins that are S cysteinylated (33). In addition, the organic peroxide sensor OhrR was inactivated by an S bacillithiolation in B. subtilis (42).Cysteine, BSH, and CoA are also dominant LMW thiols in S. aureus (52). In this study, we have investigated in more detail the extents of S thiolations and inter- and intramolecular disulfide bond formation of B. subtilis and S. aureus in response to disulfide stress. The results showed that exposure to diamide leads to S thiolations in S. aureus. Using a nonreducing/reducing sodium dodecyl sulfate (SDS) diagonal electrophoresis approach, proteins with intermolecular disulfide bonds could be distinguished from proteins with intramolecular disulfide bonds (57). The results support that the majority of reversible thiol oxidations are based on S thiolations rather than disulfide bonds between proteins. Depletion of the free cysteine pool in B. subtilis after exposure to diamide supports this finding. To assess if GSH may have a bearing on the thiol redox buffer of B. subtilis, the gshF gene of Listeria monocytogenes (gshF_Lm) was expressed in B. subtilis, enabling GSH biosynthesis (29). Although GSH production does not enhance the resistance to oxidative stress in B. subtilis, it participates in the formation of S thiolations.     

Inhibition of Selenium Metabolism in the Oral Pathogen Treponema denticola

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against Treponema denticola is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.The biological use of selenium as a catalyst, incorporated into proteins as selenocysteine, is broad. It plays an essential role in energy metabolism, redox balance, and reproduction in a variety of organisms, from bacterial pathogens to eukaryotic parasites to humans. The results of several epidemiological studies indicate that higher levels of selenium in the mammalian diet can have a negative effect on dental health (2, 17-19, 39). Although the impact of selenium is attributed to its influence on the physical properties of the enamel surface (10), the role of selenium in supporting the oral microbial community has not been studied.The oral cavity is a highly complex microbiome, with a large proportion of its residents uncharacterized due to their fastidious nature and resistance to traditional culture methods (11). Analysis of whole saliva indicates that bacterial metabolism influences the amino acid composition and indicates a role for amino acid fermentation (38). Curtis et al. demonstrated the occurrence of Stickland reactions in dental plaque (9). These reactions were first described in clostridia (35-37). They involve the coupled fermentation of amino acids in which one amino acid is oxidized (Stickland donor) and another (Stickland acceptor) is reduced (29). Treponema denticola, an established resident of the oral cavity, performs Stickland reactions via the selenoprotein glycine reductase (32). Glycine reductase is composed of a multiprotein complex that contains two separate selenoproteins, termed selenoprotein A and selenoprotein B (1, 7, 8, 15, 16). This complex of proteins converts glycine to acetyl phosphate by using inorganic phosphate and the reducing potential from thioredoxin. For the organisms that use this complex, this is a vital source of ATP. Thus far, the requirement for selenocysteine at the active site of this enzyme complex is universally conserved, even though all other selenoproteins that have been identified using computational techniques have a putative cysteine homologue (24).Treponema denticola is considered one of the primary pathogens responsible for periodontitis, a chronic inflammatory disease that is the major cause of adult tooth loss (11, 27, 33). It is the best-studied oral spirochete, commonly found with other spirochetes within the periodontal pocket. It expresses a variety of virulence factors and is capable of adhering to and penetrating endothelial cell monolayers (31). Its health impact may reach beyond the oral cavity. A recent study linked periodontitis with peripheral arterial disease and detected T. denticola, along with other periodontal pathogens, in atherosclerotic plaque (3). Sequence analysis indicates the presence of several selenoproteins in addition to glycine reductase within the genome of T. denticola (24). This organism exhibits a strict growth requirement for selenium (32).A significant literature exists that clearly demonstrates the antimicrobial activity of fluoride compounds against microorganisms associated with dental decay and periodontitis. Both sodium fluoride and stannous fluoride, as well as stannous ions alone, inhibit the growth of T. denticola (21). The inhibitory effect of stannous salts on T. denticola''s growth is unexplained. It should be noted that toothpastes containing stannous fluoride are more effective in reducing gingivitis and plaque (28, 30).Tin, as well as several other trace elements, modulates the effects of acute selenium toxicity (20). Conversely, selenium affects the activity of tin in animal models (4-6). In this study, we examine the possibility that stannous ions interfere with selenium metabolism in T. denticola.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.     

Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Mitotic centromere-associated kinesin (MCAK) plays an essential role in spindle formation and in correction of improper microtubule-kinetochore attachments. The localization and activity of MCAK at the centromere/kinetochore are controlled by Aurora B kinase. However, MCAK is also abundant in the cytosol and at centrosomes during mitosis, and its regulatory mechanism at these sites is unknown. We show here that cyclin-dependent kinase 1 (Cdk1) phosphorylates T537 in the core domain of MCAK and attenuates its microtubule-destabilizing activity in vitro and in vivo. Phosphorylation of MCAK by Cdk1 promotes the release of MCAK from centrosomes and is required for proper spindle formation. Interfering with the regulation of MCAK by Cdk1 causes dramatic defects in spindle formation and in chromosome positioning. This is the first study demonstrating that Cdk1 regulates the localization and activity of MCAK in mitosis by directly phosphorylating the catalytic core domain of MCAK.Chromosomes are properly attached to the mitotic spindles, and chromosome movement is tightly linked to the structure and dynamics of spindle microtubules during mitosis. Important regulators of microtubule dynamics are the kinesin-13 proteins (37). This kinesin superfamily is defined by the localization of the conserved kinesin core motor domain in the middle of the polypeptide (19). Kinesin-13 proteins induce microtubule depolymerization by disassembling tubulin subunits from the polymer end (6). Among them, mitotic centromere-associated kinesin (MCAK) is the best-characterized member of the family. It depolymerizes microtubules in vitro and in vivo, regulates microtubule dynamics, and has been implicated in correcting misaligned chromosomes (12, 14, 16, 24). In agreement with these observations, both overexpression and inhibition of MCAK result in a disruption of microtubule dynamics, leading further to improper spindle assembly and errors in chromosome alignment and segregation (7, 11, 15, 22, 33). The importance of MCAK in ensuring the faithful segregation of chromosomes is consistent with the observation that MCAK is highly expressed in several types of cancer and thus is likely to be involved in causing aneuploidy (25, 32).While MCAK is found both in the cytoplasm and at the centromeres throughout the cell cycle, it is highly enriched on centrosomes, the centromeres/kinetochores, and the spindle midzone during mitosis (18, 21, 36, 38). In accordance with its localizations, MCAK affects many aspects throughout mitosis, from spindle assembly and maintenance (3, 10, 36) to chromosome positioning and segregation (14, 21, 35). Thus, the precise control of the localization and activity of MCAK is crucial for maintaining genetic integrity during mitosis. Regulation of MCAK on the centromeres/kinetochores by Aurora B kinase in mitosis has been intensively investigated (1, 28, 29, 43). The data reveal that MCAK is phosphorylated on several serine/threonine residues by Aurora B, which inhibits the microtubule-destabilizing activity of MCAK and regulates its localization on chromosome arms/centromeres/kinetochores during mitosis (1, 18, 28). Moreover, in concert with Aurora B, ICIS (inner centromere KinI stimulator), a protein targeting the inner centromeres in an MCAK-dependent manner, may regulate MCAK at the inner centromeres and prevent kinetochore-microtubule attachment errors in mitosis by stimulating the activity of MCAK (27). Interestingly, hSgo2, a recently discovered inner centromere protein essential for centromere cohesion, has been reported to be important in localizing MCAK to the centromere and in spatially regulating its mitotic activity (13). These data highlight that the activity and localization of MCAK on the centromeres/kinetochores during mitosis are tightly controlled by Aurora B and its cofactors. Remarkably, MCAK concentrates at spindle poles from prophase to telophase during mitosis (18); however, only a few studies have been done to deal with that issue. Aurora A-depleted prometaphase cells delocalize MCAK from spindle poles but accumulate the microtubule-stabilizing protein ch-TOG at poles (5), implying that Aurora A might influence the centrosomal localization of MCAK in mitosis. Aurora A is also found to be important for focusing microtubules at aster centers and for facilitating the transition from asters to bipolar spindles in Xenopus egg extracts (42). In addition, it has been revealed that Ca²⁺/calmodulin-dependent protein kinase II gamma (CaMKII gamma) suppresses MCAK''s activity, which is essential for bipolar spindle formation in mitosis (11). More work is required to gain insight into the regulatory mechanisms of MCAK at spindle poles during mitosis.Deregulated cyclin-dependent kinases (Cdks) are very often linked to genomic and chromosomal instability (20). Cyclin B1, the regulatory subunit of Cdk1, is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment (2, 4). We observed that knockdown of cyclin B1 induces defects in chromosome alignment and mitotic spindle formation (N.-N. Kreis, M. Sanhaji, A. Krämer, K. Sommor, F. Rödel, K. Strebhardt, and J. Yuan, submitted for publication). Yet, how Cdk1/cyclin B1 carries out these functions is not very well understood. In this context, it is extremely interesting to investigate the relationship between the essential mitotic kinase Cdk1 and the microtubule depolymerase MCAK in human cells.