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preface nearly a decade separates this edition from its predecessor during this time progress in microbiology has been staggering in both quantity and importance whereas this is a cause for satisfaction to both practitioners and the consumers of this science it does accentuate the ancient problem of what to include in a work such as this one the solution that has presented itself is not new decide on what can be said to be the most important and central subjects to this science and ask authorities to write about the field of their expertise the topics chosen for this edition are not novel but their treatment mirrors the changes in microbiology several themes are becoming more dominant in this science among them are the following grand realizations · · · a large proportion of the biosphere is microbial and has a profound influence on nearly all aspects of this planet s metabolism including its geology cycles of matter and even its meteorology microbes have been the sole ergo the dominant force in the first 80 or so of life on earth and have played a key role in the evolution of all life forms practically all human activities have a microbial component including health and food production whereas these developments involve more and more fields of specialization microbiology has become more unified common molecular mechanisms of adhesion to surfaces quorum sensing signal transduction constructing communities or injecting proteins directly into host cells are found in dissimilar organisms that represent a broad spectrum of microbial life microbiologists now tend to speak a common language nowadays a marine microbiologist can easily talk to one studying human pathogens and a food microbiologist can converse effortlessly with one studying evolution those studying the microbial world share not just a genomic database but more profoundly the realization that microorganisms make use of similar capabilities for diverse purposes the most significant outward change in this edition has been its conversion to the web this has advantages over the printed versions including the ability to search for topics and subtopics linking to the references and avoiding the backbreaking task of carrying it around the volumes of the previous edition weighed in toto about 15 kg these would have topped 20 kg we are grateful to many colleagues who made suggestions and provided help to us special thanks go to mark jensen who made highly skillful contributions to our editing process moselio schaechter vii

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editor-in-chief moselio schaechter san diego state university san diego ca usa

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editor-in-chief professor schaechter did his graduate work at the university of kansas and the university of pennsylvania he worked on the biology of rickettsiae at walter reed army institute of research and was a postdoctoral fellow for two years in copenhagen in the laboratory of ole maaløe professor schaechter s research interest concerned various aspects of the regulation of bacterial growth he discovered the existence of polyribosomes in bacteria and was among the first to elucidate aspects of polyribosome metabolism and the role of the cell membrane in dna synthesis and chromosome segregation professor schaechter spent most of his career at tufts university in boston ma where he chaired the department of molecular biology and microbiology for 23 years since 1995 he has resided in san diego california where he teaches and continues to write books and a blog small things considered he has written nine books including several textbooks and reference works and he has served as president of the american society of microbiology and in many advisory capacities to agencies and organizations ix

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section editors sandra l baldauf uppsala university uppsala sweden john a baross university of washington seattle wa usa david c baulcombe university of cambridge cambridge uk robert haselkorn university of chicago chicago il usa david a hopwood john innes centre norwich uk john l ingraham professor emeritus university of california davis ca usa allen i laskin laskin/lawrence associates somerset nj usa bruce r levin emory university atlanta ga usa thomas m schmidt michigan state university east lansing mi usa william c summers yale university school of medicine new haven ct usa james f white jr rutgers university new brunswick nj usa xi

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applied microbiology agro/food contents agrobacterium and plant cell transformation aquaculture beer/brewing dairy products fermented foods food spoilage preservation and quality control forest products biotechnology in pulp and paper processing insecticides microbial pesticides microbial water drinking wine agrobacterium and plant cell transformation p j christie university of texas medical school at houston houston tx usa ª 2009 elsevier inc all rights reserved defining statement introduction overview of infection process ti plasmid chromosomally encoded virulence genes t-dna processing virb/d4 system a member of the type iv secretion family substrate transfer through the plant cell agrobacterium host range and genetic engineering conclusions further reading glossary autoinducer an acylhomoserine lactone secreted from bacteria which under conditions of high cell density passively diffuses across the bacterial envelope and activates transcription border sequences 25-bp direct imperfect repeats that delineate the boundaries of t-dna conjugation transfer of dna between bacteria by a process requiring cell-to-cell contact conjugative pilus an extracellular filament encoded by a conjugative plasmid involved in establishing contact between plasmid-carrying donor cells and recipient cells mobilizable plasmid conjugal plasmid that carries an origin of transfer orit but lacks genes coding for its own transfer across the bacterial envelope t-dna segment of the agrobacterium genome transferred to plant cells transconjugant a cell that has received a plasmid from another cell as a result of conjugation transfer intermediate a nucleoprotein particle composed of a single-strand of the dna destined for 1

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2 applied microbiology agro/food agrobacterium and plant cell transformation export and one or more proteins that facilitate dna delivery to recipient cells type iv secretion system a conserved family of macromolecular translocation systems evolutionarily related to conjugation systems for translocating dna or protein effector molecules between prokaryotic cells or to eukaryotic hosts abbreviations aai abc ahl cp dtr gfp ggi im mpf autoinducer atp-binding cassette acylhomoserine lactone coupling protein dna transfer and replication green fluorescent protein gonococcal genetic island inner-membrane mating pair formation nls od om sr t-dna ti tms trip t4s vbts nuclear localization sequences overdrive outer membrane substrate receptor transferred dna tumor-inducing transmembrane segments transfer dna immunoprecipitation type iv secretion virb2-interacting proteins defining statement agrobacterium tumefaciens transfers oncogenic dna tdna to susceptible plant cells causing formation of tumors called crown galls this is a multistage infection process involving sensory recognition of specific plant signals attachment to the plant host induction of a virulence regulon and t-dna processing transfer and integration into the plant genome emerged describing numerous aspects of the infection process and the myriad of ways this organism has been exploited for plant genetic engineering here i will summarize recent findings pertaining to the mechanistic details of vir gene induction t-dna processing and transfer and t-dna movement and integration in the plant host overview of infection process agrobacterium species are commonly found in a variety of environments including cultivated and nonagricultural soils plant roots and even plant vascular systems despite the ubiquity of agrobacterium species in soil and plant environments only a small percentage of isolates are pathogenic two species are known to infect plants by delivering dna to susceptible plant cells a tumefaciens is the causative agent of crown gall disease a neoplastic disease characterized by uncontrolled cell proliferation and formation of unorganized tumors agrobacterium rhizogenes induces formation of hypertrophies with a hairy-root appearance referred to as `hairy-root disease the pathogenic strains of both the species possess large plasmids ti and ri respectively that encode most of the genetic information required for dna transfer to susceptible plant cells the basic infection process is similar for both the species although the gene composition of the transferred dna differs and therefore the outcome of the infection agrobacterium has been widely viewed as the only bacterial genus capable of transferring genes to plants but in fact other members of the alphaproteobacteria can transform plants when carrying an agrobacterium ti plasmid the plant symbionts rhizobium sp ngr234 introduction agrobacterium tumefaciens is a gram-negative soil bacterium with the ability to infect plants through a process that involves delivery of a specific segment of its genome to the nuclei of susceptible plant cells the transferred dna t-dna is a discrete region of the bacterial genome delimited by 23 base pair bp direct repeats carried by the tumor-inducing ti plasmid the t-dna is important for infection because it codes for genes that when expressed in the plant cell disrupt plant cell growth and division events however this oncogenic dna can be excised from the transferred dna and replaced by virtually any gene of interest for a tumefaciens-mediated engineering of a wide array of plant species the discovery that a tumefaciens is a natural and efficient dna delivery vector spawned an entire new industry of plant genetic engineering which today has many diverse goals ranging from crop improvement to the use of plants as `pharmaceutical factories for high-level production of biomedically important proteins because of the dual importance of agrobacterium as a plant pathogen and as a dna delivery system an extensive literature has

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applied microbiology agro/food agrobacterium and plant cell transformation agrobacterium tumefaciens 3 plant cell phenolics sugars acidic ph vira virg signal recognition activation of vir genes wound nucleus vir regulon vird1 vird2 5 3 pti t-dna vire2 virf vird5 vire3 cell-cell attachment 3 5 t-dna integration virb/d4 t4s system vire2 t-dna processing vird2 t-strand translocation-competent nucleoprotein contact-dependent translocation vird2 virf vird5 vire3 figure 1 overview of agrobacterium tumefaciens infection process upon activation of the vira/virg two-component signal transduction system by signals released from wounded plant cells a single-strand transferred dna t-dna is processed from the ti plasmid and delivered as a nucleoprotein complex t-complex to plant nuclei expression of t-dna genes in the plant results in loss of cell growth control and tumor formation see text for details sinorhizobium meliloti and mesorhizobium loti were found to transfer t-dna albeit inefficiently into the chromosomes of tobacco arabidopsis and rice plants this discovery highlights the importance of the ti-plasmidencoded virulence vir genes and certain conserved chromosomal loci among these alphaproteobacteria for infection here i will focus on agrobacterium-mediated transformation as a model for understanding the requirements for interkingdom dna transfer agrobacterium-mediated transformation can be depicted as a multistage process involving 1 sensory perception of plant signals and induction of virulence genes 2 establishment of physical contact between a tumefaciens and the plant host 3 processing of t-dna and protein effectors for translocation 4 translocation across the bacterial envelope via a dedicated secretion channel 5 movement of substrates through the plant cell cytoplasm to the nucleus 6 integration of t-dna into the plant genome and 7 expression of t-dna genes see figure 1 with the exception of attachment early stages of infection are mediated by genes encoded by the ti plasmid border sequences the t-dna harbors genes that are expressed exclusively in the plant cell transcription of t-dna in the plant cell produces 39 polyadenylated rna typical of eukaryotic rna message that is translated in the cytoplasm the second region of the ti plasmid involved in infection harbors the genes responsible for sensory recognition of plant signals t-dna processing for transfer and substrate transfer across the bacterial envelope two additional regions of the ti plasmid code for functions that are not essential for the t-dna transfer process per se but are nevertheless intimately associated with the overall infection process one of these regions harbors genes involved in catabolism of novel amino acid derivatives termed opines that a tumefaciens induces plants to synthesize as a result of t-dna transfer the other encodes ti plasmid transfer functions for distributing copies of the ti plasmid and its associated virulence factors to other a tumefaciens cells by a process termed as conjugation intriguingly a novel regulatory cascade involving chemical signals released both from the transformed plant cells and the infecting bacterium serves to activate conjugative transfer of the ti plasmid among a tumefaciens cells residing in the vicinity of the plant tumor ti plasmid ti plasmids range in size from $180 to as many as 800 kilobases kb two regions of the ti plasmid contribute to infection figure 2 the first is the t-dna typically a segment of 20­35 kb delimited by 25-bp directly repeated t-dna the t-dna is delimited by dna repeats termed as border sequences figure 2 flanking one border is a

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4 applied microbiology agro/food agrobacterium and plant cell transformation auxins plant cytokinins opines lb rbo d t-dna movement vire t-dna processing/transport vird t-dna processing recruitment virc vir activator virg vir region translocation virb t-dna ti plasmid processing tra ti opine catabolism trb rep ti plasmid replication ti plasmid conjugation sensor kinase vira opine uptake degradation figure 2 regions of the ti plasmid that contribute to infection vir region and t-dna cell survival in the tumor environment opine catabolism and conjugal transfer of the ti plasmid to recipient agrobacteria tra and trb the various contributions of the vir gene products to t-dna transfer are listed t-dna delimited by 25-bp border sequences blue boxes rb right border lb left border codes for biosynthesis of auxins cytokinins and opines in the plant od overdrive sequence red box that enhances vird2-dependent processing at the t-dna border sequences sequence termed as overdrive that functions to stimulate the t-dna-processing reaction all dna between the border sequences can be excised and replaced with genes of interest and a tumefaciens will still efficiently transfer the engineered t-dna to plant cells this shows that the border sequences are the only cis elements required for t-dna transfer to plant cells additionally genes encoded on the t-dna play no role in the movement of t-dna to plant cells the t-dna genes instead code for synthesis of enzymes within transformed plant cells oncogenes synthesize enzymes involved in the synthesis of two plant growth regulators auxins and cytokinins production of these plant hormones results in a stimulation in cell division and a loss of cell growth control leading to the formation of characteristic crown gall tumors other enzymes catalyze the synthesis of novel amino acid derivatives termed as opines the ptia6 plasmid for example carries two t-dna s that code for genes involved in synthesis of octopines ­ a reductive condensation product of pyruvate and arginine other ti plasmids carry t-dnas that code for nopalines derived from -ketoglutarate and arginine and still others code for different classes of opines plants cannot metabolize opines however the ti plasmid carries opine catabolism genes that are responsible for the active transport of opines and their degradation thus providing a source of carbon and nitrogen for the bacterium the `opine concept was developed to rationalize the finding that a tumefaciens evolved as a pathogen by acquiring the ability to transfer dna to plant cells according to this concept a tumefaciens adapted a dna conjugation system for interkingdom dna transport to incite opine synthesis in its plant host the cotransfer of oncogenes ensures that transformed plant cells proliferate resulting in enhanced opine synthesis the tumor therefore is a rich chemical environment favorable for growth and propagation of the infecting a tumefaciens of further interest a given a tumefaciens strain generally catabolizes only those opines that it incites plant cells to synthesize this ensures a selective advantage of the infecting bacterium over other a tumefaciens strains that are present in the vicinity of the tumor opine catabolism the regions of ti plasmids involved in opine catabolism code for three functions related to opine catabolism the first is a regulatory function controlling expression of opine transport and catabolism genes for the octopine catabolism region of plasmid ptia6 the regulatory protein is occr a member of the family of lysr transcription factors occr positively regulates expression of the occ genes involved in octopine uptake and catabolism by inducing a bend in the dna at the occr-binding site octopine modulates occr regulatory activity by altering both the affinity of occr for its target site and the angle of the dna bend the regulatory protein for the nopaline catabolism region of plasmid ptic58 is

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applied microbiology agro/food agrobacterium and plant cell transformation 5 accr in contrast to occr accr functions as a negative regulator of acc genes involved in nopaline catabolism several other genes transcribed from a single promoter specify functions for opine transport and catabolism at the proximal end of the operon are transport genes mediating opine-specific binding and uptake typically one or more of these genes encode proteins homologous to energy-coupling proteins found associated with the socalled atp-binding cassette abc superfamily of transporters the abc transporters are ubiquitous among bacterial and eukaryotic cells and provide a wide variety of transport functions utilizing the energy of atp hydrolysis to drive the transport reaction at the distal end of the operon are genes whose products cleave opines to their parent compounds for use as carbon and nitrogen sources for the bacterium ti plasmid conjugation the ti plasmid transfer tra and trb functions direct the conjugative transfer of the ti plasmid to bacterial recipient cells figure 2 the transfer genes of conjugative plasmids code for dna-processing factors and a translocation system the ti plasmid transfer system is related in sequence and function to other plasmid transfer systems as well as dedicated protein translocation systems these systems are now classified as type iv secretion t4s systems see below a regulatory cascade activates ti plasmid transfer under conditions of high cell density figure 3 this regulatory cascade initiates when a tumefaciens imports opines released from plant cells for the octopine ptia6 plasmid occr acts in conjunction with octopine to activate transcription of the occ operon although the majority of the occ operon codes for octopine transport and catabolism functions the distal end of the occr operon encodes a gene for a transcriptional activator termed trar trar is related to luxr an activator shown nearly 20 years ago to regulate synthesis of an acylhomoserine lactone aml termed as autoinducer cells that synthesize autoinducer molecules secrete these molecules into the environment at low cell densities autoinducer is in low concentration whereas at high cell densities this substance accumulates in the surrounding environment and passively diffuses back into the bacterial cell to activate transcription of a defined set of genes in the case of a tumefaciens the autoinducer is an n-3 oxo-octonoyl l-homoserine lactone termed agrobacterium autoinducer aai aai acts in conjunction with trar to activate transcription of the ti plasmid tra genes as well as trai whose product mediates synthesis of aai therefore synthesis of trar under conditions of high cell density creates a positive-feedback loop whereby a trar­aai complex induces transcription of trai which in turn results in enhanced synthesis of more aai this regulatory cascade involving opinemediated expression of trar and trar-aai mediated expression of ti plasmid transfer genes under conditions of high cell density has the net effect of enhancing ti plasmid transfer in the environment of the plant tumor this complex regulatory system likely evolved to maximize the number of ti-plasmid-carrying bacterial cells in the vicinity of the plant wound site agrobacterium phenolics sugars low ph transformed plant cell vira virg vir gene induction t-dna t-dna transfer tra ti plasmid transfer nucleus occr opine t-dna auxins cytokinins trar tral opines opine catabolism ti trb trar aai uncontrolled cell proliferation aai tumors autoinducer aai cell growth/division figure 3 a schematic of chemical signaling events between agrobacterium and the transformed plant cell signals released from wounded plant cells initiate the infection process leading to tumor formation opines released from wounded plant cells activate opine catabolism functions for growth of infecting bacteria opines also activate synthesis of trar for autoinducer aai synthesis trar and aai at a critical concentration activate the ti plasmid conjugation functions see text for details

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6 applied microbiology agro/food agrobacterium and plant cell transformation aai-mediated activation of ti plasmid transfer is also negatively controlled for example trar activity is antagonized by two proteins tram and trlr tram interacts with the c-terminus of trar which inactivates trar and disrupts trar­dna complexes trlr is a truncated form of trar that suppresses trar activity through formation of inactive heterodimers in addition an aai signal turnover system is composed of attj a regulatory gene and attm whose product is an n-acylhomoserine lactone-lactonase that hydrolyzes the lactone ring of aai lactonase production suppresses aai-dependent expression of conjugation genes as a means of fine-tuning plasmid transfer in response to changes in cell growth and density vir genes the ti plasmid carries a $35-kb region harboring a number of operons involved in t-dna transfer figure 1 some operons have a single open reading frame while others code for up to 11 open reading frames the products of the vir region direct events within the bacterium that must precede export of a copy of the t-dna to plant cells 1 a vira/virg two-component regulatory system induces expression of the vir genes in response to perception of plant-derived signals 2 virc and vird proteins process t-dna into a nucleoprotein particle for delivery to plant nuclei and 3 a t4s system composed of virb proteins and vird4 translocates the t-dna transfer intermediate and effector proteins across the bacterial envelope figure 2 infection is initiated when bacteria sense and respond to an array of signals including specific classes of plant phenolic compounds aldose monosaccharides low po4 and an acidic ph that are present at a plant wound site figure 1 the vira/virg signal transduction system together with chve a periplasmic sugar-binding protein mediates recognition of plant phenolics and sugars vira was one of the first described of what now is recognized as a very large family of sensor kinases identified in bacteria and more recently in eukaryotic cells the members of this protein family are typically integral membrane proteins with an n-terminal extracytoplasmic domain upon sensory perception the kinase autophosphorylates at a conserved histidine residue then transferring the phosphate group to a conserved aspartate residue on the second component of this transduction pathway the response regulator the phosphorylated response regulator coordinately activates transcription of several operons whose products mediate a specific response to the inducing environmental signal for the a tumefaciens vir system the response regulator is virg and phosphorylated virg activates transcription of six essential vir operons as well as a number of other ti plasmid and chromosomally encoded operons whose products are probably important for infection of certain plant species or under certain environmental conditions the vira/virg two-component system also activates expression of the repabc genes responsible for replication of the ti plasmid plant signals thus enhance ti plasmid copy number and consequently virulence potential upon perception of environmental conditions favorable for interkingdom dna transfer vira senses most of the plant-derived signals listed above the most important signal molecules are phenols that carry an o-methoxy group the type of substitution at the para position distinguishes strong inducers such as acetosyringone from weaker inducers such as ferulic acid and acetovanillone a variety of aldose monosaccharides including glucose galactose arabinose and the acidic sugars d-galacturonic acid and d-glucuronic acid strongly enhance vir gene induction the inducing phenolic compounds as well as the monosaccharides are secreted intermediates of biosynthetic pathways involved in cell wall repair as such the presence of these compounds is a general feature of most plant wounds and likely contributes to the extremely broad host range of a tumefaciens vira functions as a homodimer and a model that vira interacts directly with inducing molecules that diffuse across the outer membrane om into the periplasm is supported by genetic experiments though direct evidence for signal binding is lacking sugarmediated inducing activity occurs via an interaction between sugars and the periplasmic sugar-binding protein chve in turn chve sugar interacts with the periplasmic domain of vira to induce a conformational change that increases the sensitivity of vira to phenolic inducer molecules a periplasmic domain of vira is also implicated in recognition of acidic ph though the physical mechanism of ph perception is unknown on the basis of recent crystallographic analysis of chey a homologue of virg phosphorylation of this family of response regulators is thought to induce a conformational change phospho-virg activates transcription of the vir genes by interacting with a cis acting regulatory sequence tncaattgaaapy called the vir box located upstream of each of the vir promoters interestingly both nonphosphorylated and phosphorylated virg bind to the vir box indicating that a phosphorylation-dependent conformation is necessary for a productive interaction with components of the transcription machinery chromosomally encoded virulence genes most studies of the a tumefaciens infection process have focused on the roles of ti plasmid genes in t-dna transfer but several essential and ancillary chromosomal genes also contribute to a tumefaciens virulence although mutations in these genes are often pleiotropic they generally function to regulate vir gene

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applied microbiology agro/food agrobacterium and plant cell transformation 7 expression or mediate attachment to plant cells this latter activity will be described in the section titled `attachment to plant cells regulators of vir gene expression at least three groups of chromosomal genes activate or repress vir gene expression as described above the periplasmic sugar-binding protein chve complexed with any of a wide variety of monosaccharides induces conformational changes in vira allowing it to interact with phenolic inducers chve mutants are severely compromised for t-dna transfer but they also show defects in chemotaxis toward sugars chve thus appears to play a dual role in the infection process by promoting bacterial chemotaxis toward nutrients and by enhancing the efficiency of opine-encoding t-dna to plant cells a second locus codes for ros a novel prokaryotic zinc finger protein that transcriptionally represses certain vir operons as described below the virc and vird operons contribute to the t-dna processing reaction although the promoters for these operons are subject to positive regulation by the vira/virg transduction system they are also negatively regulated by the ros repressor ros binds to a 9-bp inverted repeat the ros box residing upstream of these promoters in the presence of plant signals ros repression is counteracted by the vira/virg induction system but in the absence of plant signals ros binding to the virc and vird promoters prevents the t-dna-processing reaction in addition to repressing expression of t-dna processing genes in the absence of a suitable plant host ros prevents premature expression of the t-dna oncogenes in the bacterium finally a two-component regulatory system distinct from the vira/virg system senses environmental signals and mounts a behavioral response by modulating gene expression chvg is the sensor kinase and chvi is the response regulator null mutations in genes for these proteins block vir gene induction or growth of cells at an acidic ph of 5.5 the molecular basis underlying the effect of the chvg and chvi proteins on vir gene expression is presently unknown membrane cytosol and nuclear membrane the prevailing view strongly supported by molecular and genetic data is that t-dna is transferred as a nucleoprotein particle composed of a single-stranded dna molecule t-strand covalently attached to a nicking enzyme see below roles of vird2 relaxase in t-dna processing and transfer it is now widely accepted that dna-processing reactions associated with t-dna transfer are equivalent to those mediating bacterial conjugation in the generalized reaction a set of proteins termed as the dna transfer and replication dtr proteins assemble at an origin-of-transfer orit sequence to generate a nucleoprotein complex termed as the relaxosome one component of the relaxosome the relaxase cleaves and remains covalently associated with the 59 end of the dna strand destined for transfer t-strand the t-strand is unwound from its template by a strand displacement reaction generating the translocation-competent relaxase-t-strand substrate in a tumefaciens the vird2 relaxase generates nicks at orit-like sequences located in the t-dna border repeats vird2 remains covalently bound to the 59 phosphoryl end of the nicked t-dna via conserved tyrosine residue tyr-29 purified vird2 catalyzes cleavage of oligonucleotides bearing a t-dna nick site however an ancillary protein vird1 is essential for nicking in vitro when the nick site is present on a supercoiled doublestranded plasmid in addition to orit nicking the relaxase component of the conjugative transfer intermediate is thought to participate in translocation of substrate dna by supplying a signal motif recognizable by the transport machinery vird2 and other relaxases carry a motif at their extreme c termini that is devoid of secondary structure and rich in positively charged amino acids particularly arginines this motif is also present at the c-termini of protein substrates of the virb/d4 t4s system and as expected mutations in the signal motif of one such substrate virf block translocation the charged motif likely confers recognition of the substrate by the secretion channel as suggested by evidence that the vird2-t-strand complex as well as another protein substrate vire2 interact with the vird4 substrate receptor sr moreover when the c-terminal fragment of vire2 is fused to the green fluorescent protein gfp it mediates binding of the reporter protein to vird4 in living cells early studies supplied evidence for 59-39 unidirectional transfer of the t-strand which is also compatible with the notion that the relaxase serves to pilot the attached t-strand through the secretion channel t-dna processing one of the early events following attachment to plant cells and activation of vir gene expression in response to plant signals involves the processing of t-dna into a form that is competent for transfer across the bacterial cell envelope and translocation through the plant plasma

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8 applied microbiology agro/food agrobacterium and plant cell transformation roles of ancillary processing factors in t-dna processing and transfer although vird2 catalyzes nicking of t-dna substrates in vitro border cleavage in vivo requires accessory proteins including vird1 virc1 and virc2 proteins early studies showed that virc1 binds the overdrive sequence located next to the right border repeat sequences of octopine-type ti plasmids a recent quantitative analysis established that both virc1 and virc2 are required for synthesis of as many as 50 copies of the t-dna transfer intermediate per cell within a 24-h induction period a mutation in an invariant lys residue in the walker a nucleotide triphosphate binding motif of virc1 virc1k15q abolished the stimulatory effect of virc1 on t-strand production suggesting that virc1 s activity is regulated by atp binding or hydrolysis virc1 is related to the para/mind family of atpases which mediate partitioning of chromosomes and plasmids during cell division very interestingly virc1 localizes at cell poles which is also the site of virb/d4 machine assembly see below besides stimulating the conjugative processing reaction polar-localized virc1 supplies another important function to stimulate substrate translocation it recruits the vird2-t-strand nucleoprotein particle to the virb/d4 transfer machine such stimulatory functions associated with conjugation have not been described previously for other para/mind homologues but note that many mobile elements ­ both integrated and extrachromosomal ­ encode para/mind homologues in future studies it will be interesting to determine whether these proteins provide virc1-like functions to couple processed dna substrates with their cognate transfer machines virb/d4 system a member of the type iv secretion family a tumefaciens translocates the t-complex as well as effector proteins through a dedicated secretion channel assembled from 11 virb subunits and vird4 the virb proteins are termed as the mating pair formation mpf proteins and vird4 the sr also termed as the coupling protein t4cp as discussed above for the dtr-processing factors the virb and vird4 proteins are related in sequence and function to subunits of conjugation systems further underscoring the notion that a tumefaciens adapted an ancestral conjugation system to deliver effector macromolecules to plants during infection the virb/d4 system and other conjugation machines of gram-negative and -positive bacteria are members of a large family of translocation systems termed as t4s systems figure 4 in addition to the conjugation machines the t4s family encompasses two other subfamilies one termed as the `dna uptake and release systems function independently of contact with a target cell to take up dna from the extracellular milieu as exemplified by the helicobacter pylori comb competence system or to conjugation t-dna iva r388 rp4 f virb incw incp incf b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 d4 effector translocation h pylori r prowazekii brucella spp b pertussis cag virb ptl iva dna uptake/release h pylori n gonorrhoeae com iva figure 4 alignment of genes encoding related components of the t4s systems of the 11 virb proteins those encoded by virb2 through virb11 and vird4 are essential for t-complex transport to plant cells ancestrally related conjugation systems mediate interbacterial transfer of dna effector translocation systems function to secrete proteins to eukaryotic cells during the course of infection by many medically important bacterial pathogens a third subfamily of t4s systems designated as the dna uptake/release systems take up dna from the extracellular milieu or release dna to the environment.

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applied microbiology agro/food agrobacterium and plant cell transformation 9 release dna to the milieu as exemplified by a chromosomally encoded f-like transfer system carried on the gonococcal genetic island ggi of neisseria gonorrhoeae as with the conjugation machines these systems promote genetic exchange and therefore also represent potential mechanisms for transfer of survival traits during infection the third subfamily the `effector translocator systems play indispensable roles in the infection processes of many prominent pathogens of plants and mammals these machines can be viewed as `injectisomes reminiscent of the needle complexes elaborated by type iii secretion t3s machines because they deliver their substrates through direct contact with the eukaryotic target cell the list of pathogens dependent on effector translocators for disease progression includes at least two phytopathogens a tumefaciens and burkholderia cepacia plant symbionts such as s meliloti and several pathogens of mammals including h pylori legionella pneumophila and brucella and bartonella species bordetella pertussis uses an effector translocator as well but this system functions as a true exporter to deliver its toxin substrate to the extracellular milieu related systems of several additional pathogens are also implicated in the trafficking of substrates to eukaryotic cells and thus the list of t4s effector translocators continues to grow the t4s systems are classified on the basis of extensive sequence similarities with subunits of conjugation machines figure 4 although these systems are functionally versatile in terms of the substrates and target cells to which substrates are delivered they share a number of common structural and functional features that distinguish them from other known bacterial translocation systems virb/d4 type iv system machine architecture the virb/d4 system is composed of two surface structures ­ a secretion channel and a conjugative pilus figure 5 at this time there is no high-resolution structure for either structure nevertheless fairly comprehensive architectural models of the t4s system can be generated through topological structural and interaction studies of machine subunits these studies have supplied evidence for at least three stable subassemblies of virb/d4 components energy subcomplex ­ vird4 virb4 virb11 vird4 virb11 and virb4 are the three energetic components of the virb/d4 t4ss each of these subunits possesses a characteristic nucleoside triphosphate binding site walker a motif required for substrate translocation b5 b2 virb5 virb2 virb8 virb7 virb9 om virb2 virb3 virb5 virb10 b1 b10 b9 b7 b3 virb1 virb4 core complex atp vird4 b6 b4 atp atp substrate receptor cm b11 d4 figure 5 topologies structures and cellular localizations of the virb/d4 t4s subunits the agrobacterium tumefaciens virb/d4 t4s system localizes at the cell poles and is postulated to assemble as a transenvelope complex through which substrates pass to the cell surface the three atpases energize machine assembly and substrate transfer and a stable `core complex nucleates machine assembly all of the virb proteins are required to build the t pilus the virb proteins plus vird4 are required for substrate secretion the t pilus is sloughed from the cell surface and is not essential for dna or protein translocation b8 virb6 virb11 b10 p b5

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10 applied microbiology agro/food agrobacterium and plant cell transformation mutations in the walker a motifs invariably abolish substrate translocation strongly indicating that atp binding drives machine assembly or function structures of soluble domains of two vird4-like proteins have now been solved by x-ray crystallography one of trwc encoded by plasmid r388 and one of escherichi coli ftsk trwc presents as six equivalent protomers assembled as a spherical particle of overall dimensions ° ° 110 a in diameter and 90 a in height the overall structure bears a striking resemblance to the f1-atpase 3 3 heterohexamer whereas the structure of the soluble domain closely resembles dna ring helicases and other proteins such as ftsk that translocate along ss or dsdna the ftsk structure is slightly larger with an outer dia° ° meter of 120 a and a central annulus of 30 a the predicted structure is a dodecamer composed of two hexamers stacked in a head-to-head arrangement as shown by electron microscopy imaging dsdna runs through the ftsk annulus providing a structural view of a previously described atp-dependent translocase activity vird4 therefore functions as a receptor for the t-dna and protein substrates of the virb/d4 t4s system and it might also function as an inner-membrane im translocase though this needs to be explored virb11 is a member of a large family of atpases associated with systems dedicated to secretion of macromolecules purified homologues trbb trwc h pylori hp0525 and brucella suis virb11 assemble as homohexameric rings discernible by electron microscopy and the last two also by x-ray crystallography these structures present as double-stacked rings formed by the n and ° c-terminal halves and a central cavity of $50 a in diameter virb11 associates peripherally but tightly with the im of a tumefaciens and there is some evidence for atp regulation of membrane binding the role of virb11 in t4s is still fundamentally unknown virb4 subunits are large im proteins with consensus walker a and b nucleoside triphosphate-binding domains a combination of experimental studies and computer modeling has yielded a topology model depicting virb4 as predominantly cytoplasmic with possible periplasmic loops one near the n-terminus and a second just nterminal to the walker a motif as with virb11 the contribution virb4 to machine assembly and function is unknown core subcomplex ­ virb6 virb7 virb8 virb9 virb10 role in substrate translocation see below virb6 interacts with two om proteins virb7 lipoprotein and virb9 and probably also with the other virb `core subunits virb6 exerts stabilizing effects on other virb subunits and it colocalizes with vird4 and the t pilus at the cell poles of a tumefaciens the available data are consistent with a proposal that virb6 assembles as a central component of the secretion channel mediating substrate transfer across the im the virb7 lipoprotein forms a disulfide bridge with virb9 and the heterodimer sorts to the om where it exerts stabilizing effects on other machine subunits virb8 and virb10 are bitopic im subunits recently structures of periplasmic fragments of both the subunits were solved by x-ray crystallography over its length virb10 shares several structural features with tonb including a small n-terminal cytoplasmic domain a single tms a prorich region and a region of sequence conservation at the c-terminal end for tonb the pro-rich motif contributes to a rigid extended structure in the periplasm that might permit simultaneous contacts with partner subunits at the ims and oms similarly a tumefaciens virb10 interacts with the im subunits virb8 vird4 and virb4 and with the om-associated virb7­virb9 heterodimer intriguingly virb10 also functionally resembles tonb by linking energy at the im to the assembly or gating of the t4s channel for substrate translocation see below t pilus subcomplex ­ virb2 virb5 virb7 five virb proteins are implicated as forming a `core transenvelope structure on the basis of phylogenetic relationships and cell localization and protein­protein interaction data virb6 is highly hydrophobic with five predicted transmembrane segments tms and a cytoplasmic c-terminus a large central periplasmic loop designated loop p2 is now known to play an important the t4s systems involved in conjugation elaborate pili for establishing contact between plasmid-bearing donor cells and recipient cells electron microscopy studies have demonstrated the presence of long filaments approximately 10 nm in diameter on the surfaces of a tumefaciens cells induced for expression of the virb genes these filaments are absent from the surfaces of mutant strains defective in expression of one or more of the virb genes furthermore the interesting observation was made that cells grown at room temperature rarely possess pili whereas cells grown at $19 c possess these structures in abundance this finding correlates nicely with previous findings that low temperature stimulates the virb-dependent transfer of substrates to plants all of the virb proteins but not vird4 are required for the assembly of the t pilus which is composed of the virb2 pilin protein virb2 bears both sequence and structural similarity to the traa pilin subunit of the f plasmid and to the trbb subunit of plasmid rp4 virb2 like traa and rp4 is processed from a $12-kda propilin to a 7.2kda mature protein furthermore both virb2 and trbb undergo an unusual head-to-tail cyclization reaction resulting in a cyclic polypeptide that accumulates in the im virb2 polymerizes as the t pilus and virb7 lipoprotein and virb5 associate at unspecified locations along the t pilus.

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