DNA UPTAKE DURING BACTERIAL TRANSFORMATION Inês Chen and David Dubnau

Naturally competent bacteria are able to take up exogenous DNA and undergo genetic transformation. The transport of DNA from the extracellular milieu into the cytoplasm is a complex process, and requires proteins that are related to those involved in the assembly of type IV pili and type II secretion systems, as well as a DNA translocase complex at the cytoplasmic membrane. Here, we will review the current knowledge of DNA transport during transformation.

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Bacteria can acquire new genetic information by three means: conjugation, transduction and transformation. During conjugation, DNA is transferred directly from one organism to another, whereas in transduction, the DNA is carried by bacteriophages. Transformation involves the acquisition of naked DNA from the extra- cellular environment (BOX 1), and genetic competence is the ability to undergo transformation. Early experiments on transformation showed that DNA is the genetic material (BOX 2).

Since the advent of recombinant DNA technology, biologists have transformed Escherichia coli, the ‘work- horse’ of molecular biology, using procedures that alter the permeability of the cell membrane (for example, using calcium or electroporation), such that DNA can be introduced to the bacterial cell. By contrast, in this review we will discuss natural transformation, in which specialized bacterial proteins are responsible for the uptake and processing of DNA. At least 40 bacterial species, distributed through all taxonomic groups, are known to be naturally transformable1. In most of these species, genetic competence is a transient physiological state, the development of which is tightly controlled by organism-specific processes, including quorum sensing and nutritional signals2–4. It is therefore possible that many more species are competent for transformation, but the conditions in which they develop competence are still unknown5.

Transporting DNA from the extracellular milieu into the cytosolic compartment is a complex task. The incoming DNA must cross the outer membrane (in Gram-negative bacteria), the cell wall and the

cytoplasmic membrane. The outer membrane of Gram- negative bacteria represents an extra barrier for the DNA that is absent from Gram-positive bacteria, and this lends confusion to the term ‘uptake’. Uptake is defined operationally as the conversion of exogenous, DNase-sensitive DNA into a DNase-protected state. In Gram-negative bacteria, this protection can be achieved by crossing the outer membrane; by contrast, in Gram-positive bacteria, DNA uptake is synonymous with passage across the cytoplasmic membrane. Only one strand of the DNA molecule is effectively trans- ported into the cytoplasm; the other strand is degraded into nucleotides, which are released into the extracellular environment (in Gram-positive bacteria) or presumably into the periplasmic space (in Gram-negative bacteria).

Gram-positive and Gram-negative microorganisms use related proteins to import DNA (the only known partial exception, Helicobacter pylori, will be discussed later). Parts of this common competence system share homology with proteins that are involved in the assembly of type IV pili (T4P) and type II secretion systems (T2SSs), and form a structure that partially spans the cell envelope. This structure is functionally coupled to a DNA translocation complex at the cytoplasmic membrane.

We will review each of the steps of the DNA transport process and the components involved therein. Given the structural differences in the cell envelope of Gram- positive and Gram-negative bacteria, and the consequent distinct characteristics in the DNA-uptake pathways, the initial steps will be described separately. We will use Bacillus subtilis and Neisseria gonorrhoeae as prototypes for DNA transport in Gram-positive and Gram-negative

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Public Health Research Institute, Newark, New Jersey 07103, USA. Correspondence to D.D. e-mail: [email protected] doi:10.1038/nrmicro844

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outer membrane — into either the periplasmic space or specialized vesicle structures (transformasomes) that have been described in H. influenzae23.

Crossing the outer membrane: secretins. Secretins are outer-membrane proteins that are involved in the extrusion of T4P and filamentous phages, type II and type III secretion, and transformation in Gram-negative microorganisms. Secretins form stable multimeric structures, with 12 or 14 subunits, whose correct assem- bly and insertion into the outer membrane can require the presence of a specific lipoprotein, known as the pilot protein24,25. Electron microscopy shows that secretins form doughnut-like structures, with the diameter of the central cavity ranging from 6 to 8.8 nm26–28. That secretins can indeed form aqueous channels has been shown electrophysiologically29. Clearly, such large channels must be gated to preserve the integrity of the outer membrane and periplasmic compartment, and a different domain of the secretin itself probably occludes the cavity27,28.

Secretins that are involved in DNA uptake have been identified in several microorganisms. Among these, the best characterized is PilQ from N. gonorrhoeae, which also functions in pilus biogenesis30. Although PilQ is not required for pilus assembly, it is required to extrude the pilus filament across the outer membrane. The central cavity in the PilQ 12-mer, with a diameter of 6.5 nm, fits the proposed pilus fibre model (~6-nm diameter)26,31, and could easily accommodate the DNA double helix (~2.4 nm), either by itself or in a nucleoprotein com- plex. DNA uptake in N. gonorrhoeae requires the pres- ence of a DUS, so the putative DUS receptor could participate in signalling the opening of the PilQ channel during transformation; the pilus or the putative pseudopilus (see below) might also be involved. However, there is still no direct evidence that DNA passes through the secretin channel.

The type IV pilus and type II secretion To describe DNA transport during competence, we must present two closely related machines, the T4P and the T2SS. The sequence and structural similarities among the components of these three systems indicate their common origin. However, their genetic organiza- tion is not conserved, nor is there complete correspon- dence among their components, and even machines within the same class can show distinct elements.

microorganisms, respectively, as these systems are the best characterized, but we will also refer to other bacteria.

DNA binding and fragmentation in Gram-positives The binding of exogenous DNA to the surface of competent cells is the first event during transformation. ComEA, a protein with DNA-binding activity, was identified as a DNA receptor in B. subtilis (see below). Mutants lacking ComEA have a reduced ability to bind DNA6. A close orthologue of ComEA is present in Streptococcus pneumoniae, although residual DNA attachment independent of ComEA was observed in this organism7, indicating that other DNA receptor(s) might be present on the bacterial surface.

As DNA is transported linearly into the cytoplasm8, a free end must be present in the DNA molecule for transport to begin. In B. subtilis, the rate of uptake is increased by the presence of a surface endonuclease (NucA) that introduces double-stranded cleavages in the bound DNA9. In S. pneumoniae, DNA transport is preceded by the initial introduction of single-strand nicks, followed by double-strand breaks10,11, but the endonuclease that is involved in these events has not been identified.

DNA binding and uptake in Gram-negatives DNA uptake in most systems is not sequence-specific. However, in some Gram-negative microorganisms, such as Haemophilus influenzae12 and Neisseria species13, effi- cient uptake occurs only if a specific sequence is present, although the DNA binding is nonspecific. The sequence motifs that are required for efficient uptake, called DUS (DNA uptake sequences; also known as USS, for uptake signal sequences), have been identified for Neisseria sp. (5?-GCCGTCTGAA-3?)14 and H. influenzae (5?-AAGT- GCGGT-3?)15,16. The latter shares this DUS with the related bacterium Actinobacillus actinomycetemcomi- tans17. As the genomes of these bacteria are enriched in their respective DUS18–20, uptake of homospecific DNA is favoured. Specific DUS receptors on the bacterial sur- face have not yet been identified. In N. gonorrhoeae, DUS-specific binding of DNA to the cell depends on the presence of the major pilin, and can be modulated by the expression of different minor pilins21,22, which indi- cates that a DUS-binding activity is associated with the pilus or a related structure (see below). The putative receptor presumably recognizes the DUS and triggers the uptake process — the transport of DNA across the

Box 1 | Why do bacteria take up DNA and where does it come from?

Three non-mutually exclusive models can account for the evolution of DNA uptake systems72: • DNA for genetic diversity — the acquisition of potential useful genetic information, such as novel metabolic functions,

virulence traits or antibiotic resistance.

• DNA repair — environmental DNA from closely related bacteria might serve as templates for the repair of DNA damage.

• DNA as food — DNA can be used as a source of carbon, nitrogen and phosphorous90,91.

Free DNA is abundant in the environment1, as it is released from dead organisms. The development of competence in Streptococcus pneumoniae leads to lysis and DNA release from a subpopulation, providing DNA for transformation92,93. DNA can also be actively secreted by viable organisms: some strains of Neisseria gonorrhoeae use a type IV secretion system to release DNA into the medium94.

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assembled, the pilus filament extends outwards from the cytoplasmic membrane, across the peptidoglycan layer and periplasmic space, and crosses the outer membrane through a channel formed by the secretin (PilQ), assisted by its pilot protein (PilP). It has been proposed that the force from the protuding pilus fibre could be enough to open the secretin pore39. A second ATPase (PilT) is needed for twitching motility — which is caused by depolymerization and consequent retraction of the pilus filament — but is dispensable for pilus assembly40. PilF and PilT are closely related, and belong to a large family of proteins that are involved in several transport systems, called the traffic NTPases. A tip-located adhesin (PilC) seems to stabilize the pilus filament, and can also be found associated with the outer membrane.

The type II secretion system. The T2SS, also known as the secreton or main terminal branch of the general secretory pathway, mediates the translocation of pro- teins from the periplasm across the outer membrane of Gram-negative bacteria41,42. The prototypic T2SS is the pullulanase secretion apparatus of Klebsiella oxytoca, which is composed of 15 components, several of which are similar to T4P proteins (FIG. 1; TABLE 1): a traffic NTPase (PulE); a polytopic membrane protein (PulF); a secretin (PulD); a prepilin-peptidase (PulO); and several type 4 pilins (the major pilin, PulG, and the minor pilins, PulH, PulI, PulJ and PulK). The proteins from the T2SS were postulated to form a pilus-like structure, known as the pseudopilus43. Some pseudopilins can form pilus-like filaments when over- expressed44–46. These structures might correspond to abnormally long, overgrown pseudopili, and their assembly requires the other components of the T2SS.

How does the pseudopilus participate in the secre- tion of proteins across the outer membrane? It is thought that the pseudopilus spans the cell envelope, and might work as a piston, pushing specific substrates from the periplasm through the secretin pore in the outer membrane. It has been proposed that the thrust- ing pseudopilus could use the force from its elongation, which is promoted by the traffic NTPase involved in assembly (PulE in K. oxytoca). However, a second traffic NTPase (equivalent to PilT in T4P), which would medi- ate retraction of the pseudopilus, is absent from T2SSs. Perhaps pseudopilus retraction is driven by the proton motive force, which is known to be necessary for the secretion of some T2SS substrates41.

The competence pseudopilus Most competent bacteria use components that are similar to those of T4P and T2SSs to take up DNA. In competent organisms that also have T4P, there is a correlation between the presence of pili and competence47–51. However, whether pili themselves have a direct role in transformation is unclear. No interaction between DNA and the pilus or pilins has been detected52. The models for pilus structure show a cavity in the middle of the filament, but its narrow diameter (1.2 nm) and hydrophobicity argue against its use as a conduit for

Clearly, these systems have diverged and acquired unique characteristics to perform distinct tasks. The differences in these machines and their comparative analysis could prove to be as informative as their sim- ilarities. The nomenclature of the components of these systems is confusing, and we will refer only to the constituents from the prototypical systems.

The type IV pilus. T4P are long, thin appendages that are present on the surface of many Gram-negative microorganisms. T4P function in bacterial cell-to-cell interactions, adhesion to host cells and twitching motil- ity — a form of locomotion that is powered by exten- sion and retraction of the pilus filament32,33. Owing to their importance in virulence, T4P have been studied in detail. Several, if not all, proteins that participate in pilus assembly are known, but the biogenesis process itself is not yet clearly understood34. The main structural con- stituent of the pilus is type 4 pilin, which is assembled to form the pilus fibre, while a few minor pilins participate in the biogenesis process. Type 4 pilins are small pro- teins (usually <20 kDa), which are made as precursors (prepilins) that comprise a short, positively charged amino-terminal leader peptide, a hydrophobic stretch and a carboxy-terminal domain. The leader peptide and hydrophobic region sequences are relatively conserved, whereas the C-terminal region is hypervariable. The prepilins are found associated with the cytoplasmic membrane through their hydrophobic domains. They are processed by a specific enzyme that resides in the cytoplasmic membrane — the prepilin peptidase, an aspartic acid protease that cleaves the leader peptide35. Some prepilin peptidases also N-methylate the new N-terminal residue of the mature pilin, which is almost always a phenylalanine. After processing by the prepilin peptidase, which probably facilitates the translocation of mature pilins across the membrane36, the pilins are assembled into the pilus filament, which protrudes from the membrane. Two models for pilus fibres have been proposed on the basis of the X-ray crystallographic structures of two different pilins31,37. In both cases, the fibre is a helix with five subunits per turn. The core is formed by the conserved hydrophobic N-terminal domains of the mature pilins, whereas the C-terminal domains are exposed on the surface. In N. gonorrhoeae T4P38 (FIG. 1; TABLE 1), the major pilin is encoded by the pilE locus. The prepilin is processed by its peptidase (PilD). A putative ATPase (PilF) is required to assemble the pilin subunits into a filament, and might provide the energy that is necessary for polymerization. A polytopic membrane protein (PilG) also participates in pilus biogenesis. As it is Box 2 | Transformation and the discovery of DNA In 1928, Griffith reported that mixing heat-killed, virulent (capsulated, serum-resistant) pneumococci with live, non-virulent (non-capsulated, serum-sensitive) bacteria gave rise to virulent organisms, which were able to cause septicaemia in mice95. The subsequent identification by Avery, MacLeod and McCarthy of the ‘transforming principle’ — that a substance from the dead bacteria carried the information to the live, non-virulent ones — provided evidence that the genetic material is DNA96. 244 | MARCH 2004 | VOLUME 2 www.nature.com/reviews/micro R E V I E W S competence and piliation. In N. gonorrhoeae (FIG. 1; TABLE 1), these would include: the major pilin (PilE), the prepilin peptidase (PilD), the traffic NTPase (PilF), the polytopic membrane protein (PilG), the secretin (PilQ) with its pilot protein (PilP), and the pilus-stabilizing protein (PilC). We favour the existence of a competence pseudopilus as a structure that is distinct from the T4P, albeit closely related, for several reasons. Various observations indicate that the presence of functional T4P and the ability to take up DNA have different requirements — for example, the existence of pilin variants that cannot assemble efficiently into a pilus fibre, but can still support transformation, and the requirement for the presence of minor pilins for competence but not for T4P formation58–60. The major pilin should be a structural component of both T4P and the competence pseudopilus; the minor pilins could be important in determining which structure is formed. DNA53. In fact, there is considerable evidence that pili are not necessary for transformation in N. gonorrhoeae, although the expression of pilin is absolutely required54–56. Other competent organisms, such as H. influenzae or the Gram-positive bacteria B. subtilis and S. pneumoniae, require similar genes for DNA uptake, but do not possess filamentous structures that extend from the cell surface. It has been proposed that a competence pseudopilus — a structure similar to T4P — participates in the trans- port of DNA during transformation57. This putative structure would be present in both Gram-negative and Gram-positive bacteria. The proposed function of the competence pseudopilus is to bring exogenous DNA to the transport machinery that is located at the cytoplasmic membrane. In organisms with T4P, the pseudopilus would be assembled using the same components as the pilus, thereby accounting for the correlation between ComGA PulS PulD Assembly Disassembly PilC DR PulE Pullulanase PilTPilF PilP ComE PilQ a Type II secretion Klebsiella oxytoca b Type IV pilus Neisseria gonorrhoeae c Competence Neisseria gonorrhoeae d Competence Bacillus subtilis Periplasm Inner membrane ComEC ComFA Com C PilD Com GB ComEA Outer membrane PulO SecPulF ComAPilGPilD Figure 1 | Comparison of machinery required for type II secretion, type IV pilus formation and transformation in Gram-negative and Gram-positive bacteria. a | A schematic model for type II secretion, based on the pullulanase secretion system (Pul) from Klebsiella oxytoca. Not all components are represented. The pseudopilins, both major (PulG; orange) and minor (PulH,-I,-J and -K; red), are processed by the prepilin peptidase (PulO), and assembled into the pseudopilus. The polytopic membrane protein (PulF) and the traffic NTPase (PulE) participate in the process. Pullulanase (brown) is secreted into the periplasm by the Sec system, and crosses the outer membrane through a channel that is formed by the secretin (PulD), with the assistance of its pilot protein (PulS). b | A schematic model for type IV pilus formation, based on the Neisseria gonorrhoeae pilus. The major pilin (PilE; orange) and minor pilin (PilV; magenta) are processed by the prepilin peptidase (PilD), and assembled into the pilus fibre. The polytopic membrane protein (PilG) and the traffic NTPase (PilF) participate in this process. The outer-membrane/tip-located protein (PilC) stabilizes the assembled filament. The pilus crosses the outer membrane through a channel that is formed by the secretin (PilQ), with the assistance of its pilot protein (PilP). A second traffic NTPase (PilT) mediates the depolymerization of the pilus into pilin monomers and consequent retraction of the pilus. c | A schematic model for the competence pseudopilus and DNA translocase in N. gonorrhoeae. Assembly of the pseudopilus requires the same components as the type IV pilus (shown in part b). The major pilin (PilE; orange) and minor pilin (ComP; blue) are processed by the prepilin peptidase (PilD), and assembled into the pseudopilus. The polytopic membrane protein (PilG) and the traffic NTPase (PilF) participate in this process, as well as PilC (not shown). The specific sequence in the exogenous DNA that is required for efficient uptake is recognized by its postulated, but as-yet-unidentified, receptor (DR). The incoming DNA is transported across the outer membrane through a channel that is formed by the secretin (PilQ), with the assistance of its pilot protein (PilP). The periplasmic DNA-binding protein (ComE) is involved in uptake, and delivers the DNA to the channel at the cytoplasmic membrane (ComA). One strand enters the cytosol; the other is degraded and the degradation products are released into the periplasmic space. d | A schematic model for the competence pseudopilus and DNA translocase in Bacillus subtilis. The major pseudopilin (ComGC; orange) and minor pseudopilins (ComGD, -GE and -GG; blue) are processed by the prepilin peptidase (ComC), and assembled into the pseudopilus. The polytopic membrane protein (ComGB) and the traffic NTPase (ComGA) participate in this process. The pseudopilus allows the exogenous DNA to access its membrane-bound receptor (ComEA), which delivers the bound DNA to the channel at the cytoplasmic membrane (ComEC). An ATP-binding protein (ComFA) is involved in DNA transport across the membrane. One strand enters the cytosol, while the other is degraded and the degradation products are released into the extracellular milieu. NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | MARCH 2004 | 245 R E V I E W S receptor. Second, PilT orthologues are only present in bacteria with T4P; other organisms have a single competence traffic NTPase, which is presumably involved in assembly of the pseudopilus. It is conceivable that this single traffic NTPase could function in both assembly and disassembly of the pseudopilus; another possibility is that the retraction of the pseudopilus is powered by other means, such as the proton motive force, which participates in T2SS function and has also been implicated in DNA uptake in B. subtilis 66. However, in a P. stutzeri strain expressing a pilin mutant that could not be assembled into a fibre, PilT was not required for transformation65. This indicates that PilT, which has a negative effect on pilus assembly39,67, might not be directly involved in the uptake process; instead, PilT might be required for competence only because it allows the pseudopilus to compete for the common assembly machinery that is shared with T4P. In the absence of PilT, the formation of T4P would be favoured, to the detriment of pseudopilus assembly, and competence would be abolished. The membrane translocation machinery The translocation of DNA across the cytoplasmic membrane has been studied most in B. subtilis, where it requires three components: the DNA receptor ComEA, the permease/channel protein ComEC and the ATP- binding protein ComFA. Other organisms seem to use the same basic components (TABLE 1). The putative com- plex in B. subtilis is classified by the Transport Protein Database as a DNA translocase (TC 3.A.11.1.1)68. It has recently been proposed that the DNA translocase is actually an atypical ATP-binding cassette (ABC) transporter, the substrate of which is the incoming DNA during transformation (I. Draskovic and D.D., unpublished observations). ABC transporters constitute one of the largest families of proteins and are present in all organisms, both the prokaryotes and the eukaryotes69. They can be exporters or importers, using energy from ATP hydrolysis to translocate solutes across the cytoplasmic membrane. The translocation component consists of two intracellu- lar nucleotide-binding domains and two transmembrane domains, which can assume different arrangements, but which contain characteristic signature motifs. The substrate-binding protein, a component from ABC transporters that is involved in solute uptake, is located in the periplasm (in Gram-negative bacteria) or is anchored to the membrane (in Gram-positive bacteria and Archaea). The DNA receptor. ComEA is a protein that is required for B. subtilis transformation, and mutants that lack ComEA are impaired in their ability to bind DNA6. The ComG proteins, which form the putative competence pseudopilus (see above), are required to provide access for the incoming DNA to ComEA, which is bound to the membrane through its N-terminal region. The C-terminus of ComEA contains the DNA-binding domain, which is composed of helix–hairpin–helix motifs that interact with double-stranded DNA without Supporting this hypothesis, two minor pilins have antagonistic effects in competence in N. gonorrhoeae : the expression of PilV inhibits transformation22, whereas transformation is enhanced by ComP58. A minor pilin with a negative effect on transformation (PilAII) has also been identified in Pseudomonas stutzeri 61. Microorganisms that lack T4P have an apparatus that is presumably devoted to assembly of the pseudopilus. In B. subtilis 62 (FIG. 1; TABLE 1), this appa- ratus includes the ComG proteins: a traffic NTPase (ComGA), a polytopic membrane protein (ComGB), four pseudopilins (one major, ComGC, and 3 minor, ComGD, ComGE and ComGG), as well as a protein with no known orthologues in other systems (ComGF). Other proteins are involved in the processing of the pseudopilins in B. subtilis: the prepilin peptidase (ComC) cleaves their N-terminal leader peptides, and a protein oxidoreductase pair (BdbD and BdbC) intro- duces an intramolecular disulphide bond into the major pseudopilin, ComGC, which is essential for its stability63. The ComG proteins are required for DNA binding to the competent cell, but the pseudopilins themselves do not show DNA-binding activity64. The ComG proteins are thought to modify the cell wall, perhaps by creating a channel, which allows the exogenous DNA to gain access to the membrane- anchored receptor ComEA. In fact, the ComG proteins are not required for DNA binding if the cell wall is removed64. Furthermore, the major pseudopilin (ComGC) can be found in the membrane fraction, as well as associated with the cell wall. Like other traffic NTPases, ComGA contains a nucleotide-binding site with Walker A and B boxes, and other conserved motifs; a mutation in the Walker A box leaves the cells incapable of binding DNA. ComGA might provide the energy that is necessary for pseudopilus assembly through ATP hydrolysis62. In Gram-positive bacteria, the competence pseudopilus would span the thick cell wall, whereas in Gram-negative bacteria it would extend across the periplasmic space and peptidoglycan layer, bridging the outer and inner membranes. How would it partici- pate in DNA uptake? As stated above, no interaction between DNA and pilins or pseudopilins has been detected. In B. subtilis, the pseudopilus could have a passive role, forming a structure across the cell wall that allows exogenous DNA to contact its membrane- bound receptor. However, in organisms with T4P, the traffic NTPase that mediates pilus retraction (PilT) is required for DNA uptake40,65, which indicates that the pseudopilus could retract and pull DNA into the cell from the bacterial surface, facilitating the interaction of the incoming DNA with its receptor (see below). In Gram-negative bacteria, the retraction of the pseudopilus would mediate passage of the DNA across the secretin channel. Despite its appeal, this hypothesis has several short- comings. First, it would involve DNA binding to the pseudopilus itself or to a protein that is associated with it, neither of which have been documented. However, in N. gonorrhoeae, this protein could be the elusive DUS 246 | MARCH 2004 | VOLUME 2 www.nature.com/reviews/micro R E V I E W S DNA and trigger the opening of the secretin channel, thereby allowing the passage of DNA — which could be mediated by ComE. So, ComE could link the com- petence pseudopilus (which also participates in uptake) with the transport machinery at the inner membrane. The DNA channel across the cytoplasmic membrane. ComEC is a polytopic membrane protein that is necessary for B. subtilis transformation. ComEC is dispensable for DNA binding, but is essential for DNA transport, and it has been proposed that this protein forms an aqueous channel across the cyto- plasmic membrane72. ComEC is present as a homod- imer, with five transmembrane domains per subunit, and contains a signature motif that is characteristic of ABC transporter permeases — the binding-protein domain (BPD). Mutation of a residue in this region reduces the level of transformation (I. Draskovic and D.D., unpublished observations). Orthologues of B. subtilis ComEC have been iden- tified in other competent organisms and its role in transformation has been verified in several cases7,71,73,74. In N. gonorrhoeae, the orthologous protein (ComA) is not necessary for DNA uptake; mutants that lack ComA retained the incoming DNA as a double-stranded molecule in the periplasmic space75. The ATP-binding protein. B. subtilis ComFA is a membrane-associated protein that is required for competence; its mutation leads to a 1,000-fold reduc- tion in transformation frequency, … Read more Applied Sciences Architecture and Design Biology Business & Finance Chemistry Computer Science Geography Geology Education Engineering English Environmental science Spanish Government History Human Resource Management Information Systems Law Literature Mathematics Nursing Physics Political Science Psychology Reading Science Social Science Home Homework Answers Blog Archive Tags Reviews Contact twitterfacebook Copyright © 2021 SweetStudy.com