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.     

Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of Cronobacter Genomospecies (Enterobacter sakazakii) in Powdered Infant Formula

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.Cronobacter strains were originally described as Enterobacter sakazakii (12), but they are now known to comprise a novel genus consisting of six separate genomospecies (20, 21). These opportunistic pathogens are ubiquitous in the environment and various types of food and are occasionally found in the normal human flora (11, 12, 16, 32, 47). Based on case reports, Cronobacter infections in adults are generally less severe than Cronobacter infections in newborn infants, with which a high fatality rate is associated (24).The ability to detect Cronobacter and trace possible sources of infection is essential as a means of limiting the impact of these organisms on neonatal health and maintaining consumer confidence in powdered infant formula (PIF). Conventional methods, involving isolation of individual colonies followed by biochemical identification, are more time-consuming than molecular methods, and the reliability of some currently proposed culture-based methods has been questioned (28). Recently, several PCR-based techniques have been described (23, 26, 28-31, 38). These techniques are reported to be efficient even when low levels of Cronobacter cells are found in a sample (0.36 to 66 CFU/100 g). However, PCR requires DNA extraction and does not allow direct, in situ visualization of the bacterium in a sample.Fluorescence in situ hybridization (FISH) is a method that is commonly used for bacterial identification and localization in samples. This method is based on specific binding of nucleic acid probes to particular DNA or RNA target regions (1, 2). rRNA has been regarded as the most suitable target for bacterial FISH, allowing differentiation of potentially viable cells. Traditionally, FISH methods are based on the use of conventional DNA oligonucleotide probes, and a commercial system, VIT-E sakazakii (Vermicon A.G., Munich, Germany), has been developed based on this technology (25). However, a recently developed synthetic DNA analogue, peptide nucleic acid (PNA), has been shown to provide improved hybridization performance compared to DNA probes, making FISH procedures easier and more efficient (41). Taking advantage of the PNA properties, FISH using PNA has been successfully used for detection of several clinically relevant microorganisms (5, 15, 17, 27, 34-36).     

High Abundance of Ammonia-Oxidizing Archaea in Coastal Waters,Determined Using a Modified DNA Extraction Method

Molecular characterizations of environmental microbial populations based on recovery and analysis of DNA generally assume efficient or unbiased extraction of DNA from different sample matrices and microbial groups. Appropriate controls to verify this basic assumption are rarely included. Here three different DNA extractions, performed with two commercial kits (FastDNA and UltraClean) and a standard phenol-chloroform method, and two alternative filtration methods (Sterivex and 25-mm-diameter polycarbonate filters) were evaluated, using the addition of Nitrosopumilus maritimus cells to track the recovery of DNA from marine Archaea. After the comparison, a simplified phenol-chloroform extraction method was developed and shown to be significantly superior, in terms of both the recovery and the purity of DNA, to other protocols now generally applied to environmental studies. The simplified and optimized method was used to quantify ammonia-oxidizing Archaea at different depth intervals in a fjord (Hood Canal) by quantitative PCR. The numbers of Archaea increased with depth, often constituting as much as 20% of the total bacterial community.Efficient DNA extraction from environmental samples is fundamental to many culture-independent characterizations (10). Thus, there was an early and concerted effort to establish appropriate methods of DNA extraction from different types of environmental samples (14, 19, 25, 30, 34, 43, 47). DNA extraction efficiency is particularly important for quantitative PCR (qPCR), because poor DNA extraction efficiency results in the underestimation of gene copy numbers in the samples examined (6, 42).Most methodological developments addressed DNA extraction from soil and sediment samples, with fewer comparative studies of the efficiency of collection and extraction from water samples (4, 13, 40). In part, a methodological focus on soils reflected the simplicity of filtration to collect aquatic populations and the generally good recovery of DNA from the Gram-negative bacteria making up a significant fraction of aquatic communities. However, small Archaea are now known to constitute a substantial fraction of the prokaryotic populations in marine and terrestrial systems (2, 7, 9, 20, 26, 31, 33, 45). Since the archaeal cell wall and membrane structures are distinct from those of bacteria, there is no assurance that commonly used extraction methods are adequate. With increasing reliance on commercially available bead-beating-type DNA extraction kits, these methods are now often used for different water samples (1, 5-7, 14, 19, 36). Although most protocols incorporate mechanical disruption to ensure more-uniform extraction than is possible by using methods that rely entirely on enzymatic digestion and/or chemical disruption (4, 13, 40), the suitability of these protocols for the concerted analysis of archaeal and bacterial populations has not been fully evaluated.In the studies reported here, the recently isolated marine archaeon Nitrosopumilus maritimus strain SCM1 (22) was therefore used as a reference standard for evaluation of the commonly employed DNA extraction methods by using qPCR. This archaeon was then used as a reference for the development of a simple, rapid, and efficient method of extracting DNA from both archaeal and bacterial cells. The modified protocol was subsequently employed to characterize the vertical distribution of ammonia-oxidizing Archaea in a fjord (Hood Canal) in Puget Sound (Washington State), revealing a high fractional representation of Archaea relative to Bacteria not observed previously in coastal waters.     

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.The genera Thermoanaerobacter and Thermoanaerobacterium contain bacteria which are thermophilic, obligate anaerobes that specialize in polysaccharide and carbohydrate fermentation, producing primarily l-lactic acid, acetic acid, ethanol, CO₂, and H₂ (24, 27, 49). Taxonomically, they are distinguished from other anaerobic thermophilic clostridia by the ability to reduce thiosulfate to hydrogen sulfide or elemental sulfur (21). The majority of characterized Thermoanaerobacter and Thermoanaerobacterium strains have been isolated from hot springs and other thermal environments (20-22, 38, 47); however, they have also been isolated from canned foods (4, 10), soil (48), paper mills and breweries (41, 43), and deep subsurface environments (5, 13, 35), suggesting a somewhat ubiquitous environmental presence.Representatives of the Thermoanaerobacter and Thermoanaerobacterium genera have been considered for biotechnological applications, such as conversion of lignocellulosic biomass to ethanol (8, 27) or other fuels and chemicals (3, 24). However, the branched fermentation pathways of these organisms generally require modification for industrial application. Several studies have investigated manipulating bioprocess and growth conditions to alter end product ratios and yields, but this has not resulted in reliable conditions to maximize the yield of a single end product (18, 25). Genetic engineering is likely necessary for commercial application of Thermanaerobacter or Thermoanaerobacterium species (26, 27, 44). As genetic systems for these bacteria have emerged (28, 45), increased product yields have been demonstrated by gene knockout of l-lactate dehydrogenase (9, 14), phosphotransacetylase and acetate kinase (40), and hydrogenase (39). Despite this recent progress, genetic transformation is still considered the greatest barrier for engineering these organisms (44).In contrast, some of the bacteria most amenable to genetic manipulation are those exhibiting natural competence; for example, work with the naturally competent Streptococcus pneumoniae first established DNA as the molecule containing inheritable information (42). Naturally competent organisms are found in many bacterial phyla, although the overall number of bacteria known to be naturally competent is relatively small (16).The molecular mechanisms of natural competence are often divided into two stages: early-stage genes that encode regulatory and signal cascades to control competence induction, and late-stage genes that encode the machinery of DNA uptake and integration (16). The Gram-positive late-stage consensus mechanism for DNA uptake and assimilation, elucidated primarily through work with Bacillus subtilis, occurs through several molecular machinery steps. First, DNA is believed to interact with a type IV pilus (T4P) or pseudopilus that brings it into close proximity of the cell membrane. The precise mechanism of this phenomenon is unclear; although components of the T4P in both Gram-positive and Gram-negative bacteria have been shown to bind DNA (7, 19), in specific studies, a full pilus structure has been either not observed or shown not to be essential during natural competence (6, 36). Two proteins, ComEA and ComEC, are then involved in creation and transport of single-stranded DNA across the membrane, where it is subsequently bound by CinA-localized RecA and either integrated into the genome or replicated at an independent origin, as for plasmid DNA (6).Here, we report that several Thermoanaerobacter and Thermoanaerobacterium strains are naturally competent, characterize growth conditions conducive to natural competence, and identify genes in Thermoanaerobacterium saccharolyticum JW/SL-YS485 required for competence exhibition.     

Virus-Bacterium Coupling Driven by both Turbidity and Hydrodynamics in an Amazonian Floodplain Lake

The importance of viruses in aquatic ecosystem functioning has been widely described. However, few studies have examined tropical aquatic ecosystems. Here, we evaluated for the first time viruses and their relationship with other planktonic communities in an Amazonian freshwater ecosystem. Coupling between viruses and bacteria was studied, focusing both on hydrologic dynamics and anthropogenic forced turbidity in the system (Lake Batata). Samples were taken during four hydrologic seasons at both natural and impacted sites to count virus-like particles (VLP) and bacteria. In parallel, virus-infected bacteria were identified and quantified by transmission electron microscopy (TEM). Viral abundance ranged from 0.5 × 10⁷ ± 0.2 × 10⁷ VLP ml⁻¹ (high-water season, impacted site) to 1.7 × 10⁷ ± 0.4 × 10⁷ VLP ml⁻¹ (low-water season, natural site). These data were strongly correlated with the bacterial abundance (r² = 0.84; P < 0.05), which ranged from 1.0 × 10⁶ ± 0.5 × 10⁶ cells ml⁻¹ (high water, impacted site) to 3.4 × 10⁶ ± 0.7 × 10⁶ cells ml⁻¹ (low water, natural site). Moreover, the viral abundance was weakly correlated with chlorophyll a, suggesting that most viruses were bacteriophages. TEM quantitative analyses revealed that the frequency of visibly infected cells was 20%, with 10 ± 3 phages per cell section. In general, we found a low virus-bacterium ratio (<7). Both the close coupling between the viral and bacterial abundances and the low virus-bacterium ratio suggest that viral abundance tends to be driven by the reduction of hosts for viral infection. Our results demonstrate that viruses are controlled by biological substrates, whereas in addition to grazing, bacteria are regulated by physical processes caused by turbidity, which affect underwater light distribution and dissolved organic carbon availability.Viruses are the most abundant and dynamic components of the aquatic microbial community (6, 31, 32). Viruses influence many biogeochemical and ecological processes, including nutrient cycling, system respiration, particle-size distribution, bacterial and algal biodiversity, species distribution, algal blooms, and genetic transfer between microorganisms (21, 49). In addition, viruses play a major role in aquatic microbial food webs by releasing carbon trapped in host cells to the dissolved organic carbon (DOC) pool and ultimately back to the bacterial community (11, 21). The action of viruses is an important mechanism of bacterial regulation in aquatic ecosystems, acting directly on bacterial populations and indirectly on bacterial diversity by decreasing the density of dominant bacterial species (31). Studies based on viral decay rates and electron microscopy analyses have shown that viruses can cause up to 40% of bacterial mortality and more than 10% of phytoplankton mortality in aquatic systems (11, 22, 48, 50, 54, 55). It also has been suggested that viral lysis and protistan grazing cause similar bacterial mortality in aquatic ecosystems (22, 40).Several environmental factors, including solar radiation and temperature, can influence viral abundance. Exposure to solar radiation decreases viral abundance in aquatic ecosystems, while low temperatures decrease their virulence (33, 56). However, the majority of the studies on virus ecology have been performed in temperate or polar regions, where seasonal changes in solar radiation and water temperature are more pronounced (26, 30, 32). Viral abundances have been little investigated in tropical aquatic ecosystems (6, 39) and particularly in the Amazonian region, where the abundance and activity of aquatic viruses have not been studied.The greatest watershed in the world is located in the Amazonian region. It is composed of clear-water, black-water, and turbid freshwater ecosystems, which are seasonally influenced by the flood pulse. The hydrologic pulse is characterized by a pronounced change in water level, defining the flood seasons. Nutrient sources and stocks and species dynamics vary according to the water level (25). During the high-water season (flood season), the tight connection between terrestrial and aquatic environments results in an increase in allochthonous DOC input and the dilution of inorganic nutrients and organisms. During the low-water season, there is an increase in nutrient concentrations, organism abundances, and the importance of autochthonous DOC. These seasonal changes differently impact ecosystem functions and aquatic community dynamics (5, 9, 18). For instance, bacterioplankton abundance is less changeable than phytoplankton abundance throughout the hydrological cycle due to the alternative sources of DOC (allochthonous in the high-water period and autochthonous in the low-water period) for bacterial communities (5, 24).Lake Batata is a clear-water Amazonian floodplain lake located in the watershed of the Trombetas River, a tributary of the Amazon River. As a clear-water Amazonian ecosystem, it contains low concentrations of suspended particles and inorganic nutrients (47). Lake Batata is distinct because it was impacted by bauxite tailings for 10 years (1979 to 1989), affecting 30% of the lake''s area. The tailings caused a huge increase in turbidity; large amounts of tailings settled on the sediment surface and often are resuspended by physical mixing or biotic movements (28). The presence of tailings resulted in a clear spatial variation in the lake, forming impacted and natural sites. Furthermore, tailing particles can directly act as a substrate for attaching bacteria and also can adsorb DOC (5).Previous studies on bacterio-, phyto-, and zooplankton communities have shown that flood pulse acts as the primary driver of plankton community structure in Lake Batata (5, 9, 24). Bauxite tailings also affect microbial processes in impacted sites, such as bacterial growth and production (5), photosynthesis rates and primary production (43), or the availability of food for zooplankton (8). However, there still is no evidence indicating a complementary (flood pulse and forced turbidity) effect among these factors in any microbial community. Based on published data, we assume that (i) bacterioplankton abundance is less changeable through the hydrologic cycle than phytoplankton abundance (5, 24), and (ii) tailing particles can act as a substrate for attaching bacteria and also can adsorb organic matter, which is controlled by the flood pulse (5). Therefore, we hypothesized that the relationship between viruses and bacteria in Lake Batata is modulated by a synergistic effect between the hydrological cycle and turbidity.     

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject''s stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.Helicobacter pylori, a gram-negative bacterium, is remarkable for its ability to persist in the human stomach for decades. Colonization with H. pylori increases risk for peptic ulcer disease and gastric adenocarcinoma (53, 70) and elicits a vigorous immune response (15). The persistence of H. pylori occurs in a niche in the human body previously considered inhospitable to microbial colonization: the acidic stomach replete with proteolytic enzymes.H. pylori strains exhibit substantial genetic diversity, including extensive variation in the presence, arrangement, order, and identity of genes (2, 4-7, 25, 51, 74). Furthermore, analyses of multiple single-colony H. pylori isolates from separate stomach biopsy specimens of individual patients have demonstrated diversity, both within hosts (27, 65), and over time (36). The mechanisms that generate H. pylori genetic diversity may be among the factors that enable persistence in this environment (3, 28).While the natural ability of H. pylori for transformation and recombination may explain some of the intra- and interhost genetic variation observed in this bacterium (43), point mutations and interspecies recombination alone are not sufficient for explaining the extent of the variation in H. pylori (14, 32). The initial genomic sequencing of H. pylori strains 26695 and J99 (6, 72) revealed large amounts of repetitive DNA (1, 59). DNA repeats in bacteria are associated with mechanisms of plasticity, such as phase variation (49, 67); slipped-strand mispairing (41, 46); and increased rates of recombination, deletion, and insertion (17, 60, 62). Because many of the recombination repair and mismatch repair mechanisms common in bacteria are absent or modified in H. pylori (28-30, 56, 76), this organism may be particularly susceptible to the diversifying effects of repetitive DNA. In fact, loci in the H. pylori genome containing repetitive DNA have been shown to exhibit extensive inter- and intrahost variation (9, 10, 28, 37).We hypothesized that identification of repetitive DNA hotspots in H. pylori would allow the recognition of genes whose variation could aid in persistence. To examine this hypothesis, we conducted in silico analyses to identify open reading frames (ORFs) enriched for DNA repeats and then used a combination of sequence analyses and immunoassays to examine the patterns associated with the specific repetitive DNA observed. Our approach led to the realization that a previously identified H. pylori-specific gene family (19, 52) exhibits extensive genetic variation at multiple levels.     

Assessment of Bias Associated with Incomplete Extraction of Microbial DNA from Soil

DNA extraction bias is a frequently cited but poorly understood limitation of molecular characterizations of environmental microbial communities. To assess the bias of a commonly used soil DNA extraction kit, we varied the cell lysis protocol and conducted multiple extractions on subsamples of clay, sand, and organic soils. DNA, as well as bacterial and fungal ribosomal gene copies as measured by quantitative PCR, continued to be isolated in successive extractions. When terminal restriction fragment length polymorphism was used, a significant shift in community composition due to extraction bias was detected for bacteria but not for fungi. Pyrosequencing indicated that the relative abundances of sequences from rarely cultivated groups such as Acidobacteria, Gemmatimonades, and Verrucomicrobia were higher in the first extraction than in the sixth but that the reverse was true for Proteobacteria and Actinobacteria. This suggests that the well-known phylum-level bacterial cultivation bias may be partially exaggerated by DNA extraction bias. We conclude that bias can be adequately reduced in many situations by pooling three successive extractions, and additional measures should be considered when divergent soil types are compared or when comprehensive community analysis is necessary.The vast majority of soil bacteria (1, 7, 27) and fungi (13, 29) cannot be cultured via traditional laboratory techniques and must be identified using molecular methods. Successful characterization of microbial communities is therefore often dependent on DNA that is extracted from the environment. However, extraction of high-quality DNA from soil can be problematic (8, 11, 22, 26). Commercial DNA extraction kits are now commonly used in the assessment of taxonomic and functional diversity, community composition, and population abundance (e.g., references 19, 21, 23, 25, and 31). Studies comparing various kits (18, 32) or comparing commercial kits to other methods (2, 10, 24) have shown that DNA yield and purity vary depending on methodology and soil type. While these comparative studies are valuable, it is still unclear to what extent these protocols yield genomic DNA representative of the microbial community found within soil.Our objective in this study was to optimize and assess the bias of a widely used commercial soil DNA extraction kit. We hypothesized that cell lysis would be enhanced and DNA would be removed from adsorption sites by conducting multiple extractions on a single sample, thereby increasing genomic DNA yield and obtaining a more complete survey of microbial taxa. This hypothesis was tested by (i) varying the extraction protocol and measuring DNA yield for three soils with differing characteristics and (ii) examining extraction bias in the genomic DNA obtained from successive extractions by using an improved method. Analytical replicates rather than biological replicates were used in order to focus strictly on variation and bias introduced through methodology, although multiple soil types were analyzed to determine whether biases detected were consistent.     

Phylogenetic Analysis Indicates Evolutionary Diversity and Environmental Segregation of Marine Podovirus DNA Polymerase Gene Sequences

The distribution of viral genotypes in the ocean and their evolutionary relatedness remain poorly constrained. This paper presents data on the genetic diversity and evolutionary relationships of 1.2-kb DNA polymerase (pol) gene fragments from podoviruses. A newly designed set of PCR primers was used to amplify DNA directly from coastal sediment and water samples collected from inlets adjacent to the Strait of Georgia, British Columbia, Canada, and from the northeastern Gulf of Mexico. Restriction fragment length polymorphism analysis of 160 cloned PCR products revealed 29 distinct operational taxonomic units (OTUs), with OTUs within a site typically being more similar than those among sites. Phylogenetic analysis of the DNA pol gene fragments demonstrated high similarity between some environmental sequences and sequences from the marine podoviruses roseophage SIO1 and cyanophage P60, while others were not closely related to sequences from cultured phages. Interrogation of the CAMERA database for sequences from metagenomics data demonstrated that the amplified sequences were representative of the diversity of podovirus pol sequences found in marine samples. Our results indicate high genetic diversity within marine podovirus communities within a small geographic region and demonstrate that the diversity of environmental polymerase gene sequences for podoviruses is far more extensive than previously recognized.Marine viruses are the most abundant (41) and diverse (2, 6) biological entities in the ocean. They affect community composition by causing the lysis of specific subsets of the microbial community (22, 28, 46, 47) and, by killing numerically dominant host taxa, may influence species evenness and richness (24, 28, 43, 50). Despite the abundance of bacteriophages in marine systems and their important roles in marine microbial composition, little is known about the distribution and diversity of specific groups of marine viruses. However, most marine bacteriophage isolates are tailed phages (3) belonging to the order Caudovirales (27), which comprises the families Myoviridae, Podoviridae, and Siphoviridae.Podoviruses are classified into several groups (e.g., T7-like, P22-like, and phi-29-like) based on genome size, genome arrangement, and shared genes and can be readily isolated from seawater (11, 16, 42, 45). Genomic analysis of roseophage SIO1 (33), cyanophage P60 (7), vibriophage VpV262 (21), and cyanophage PSSP7 (40) suggests that many of the isolates are T7-like. Despite the apparently wide distribution of podoviruses in the sea, and their potential importance as agents of microbial mortality, there has been little effort to explore their diversity.Sequence analysis of representative genes is one approach that has been used to examine the genetic diversity of specific groups of marine viruses. For example, homologues for structural genes (g20 and g23) found in T4-like phages are found in some marine myoviruses (18, 20) and have been used to examine the distribution, diversity, and evolutionary relationships among marine myoviruses (12, 14, 17, 37, 38, 49). Other studies have used DNA polymerase (pol) to examine the diversity of viruses infecting eukaryotic phytoplankton (8, 38) and have shown that phylogenies constructed with this gene are congruent with established viral taxonomy (9, 36, 37).Although it is not universally present, family A DNA pol is a good target for examining the diversity of podoviruses (4). Our study presents a newly designed set of PCR primers that amplify a longer fragment of the DNA polymerase from a much larger suite of podoviruses and shows that the diversity within marine podoviruses as revealed by DNA pol sequences is far greater than previously realized.     

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).