Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA

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Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

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One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

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Bacterial biofilms : A common cause of persistent infections

▪ Abstract Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that ba...

Quorum Sensing in Bacteria

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

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The complete genome sequence of the gastric pathogen Helicobacter pylori

In a process called quorum sensing, bacteria produce and secrete certain signaling compounds (called autoinducers) that bind to receptors on other bacteria and activate transcription of certain genes. A clever genetic selection yields a new quorum-sensing transcriptional regulator that marches to the beat of a different drummer.

Bacterial evolution by intelligent design.

The Cell-Cell Communication System of Agrobacterium Tumefaciens

OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors OccR also functions as an autorepressor in the presence or absence of octopine OccR binds to a site between occQ and occR in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression The mutations in these mutants spanned most of the length of the protein Two additional positive-control mutants were isolated using site-directed mutagenesis Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation

Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend.

It has been 100 years since Agrobacterium tumefaciens was demonstrated to cause crown gall tumors at plant wound sites. This chapter first presents background information relevant to quorum sensing in A. tumefaciens and then focuses on our current knowledge of the molecular biology of the TraR-TraI system. Regulation of TraR activity is complex and occurs at the levels of transcription, protein folding, resistance to proteolysis, and the formation of quaternary complexes with other TraR subunits or with two different antiactivators of TraR. Control of traR expression by opines therefore has evolved independently in these two types of Ti plasmids. The genes of the arc operon and occ operon are not similar, except for traR. LysR and AccR are also dissimilar, as OccR is a LysR-type transcriptional activator that binds to promoter DNA both in the presence and absence of the inducing signal. Overproduction of TraR can fully overcome inhibition, suggesting that TraM acts by making stoichiometric contacts with TraR. Direct interactions were confirmed by yeast two-hybrid assays and by far Western immunoblots. The author recently used gene arrays to profile the TraR transcriptome and found that all induced genes were Ti plasmid-encoded. These genes include the tra and trb genes, which are involved in conjugal transfer; the rep genes, which are required for vegetative replication and plasmid partitioning into daughter cells; and traM.

Cell-Cell Signaling within Crown Gall Tumors

TwopKM101-encoded loci (designated kilA andkiLB) havebeenidentified whichelaborate products that are potentially lethal tothebacterial cell. Thelethal effects ofeachofthese geneproducts isinhibited bytwoother plasmid-encoded loci, designated korAandkorB(for kiloverride). BothkorAandkorBarerequired tocontrol thelethality ofeither kit gene.InthepresenceofkorAandkorBbothkil geneshaveother phenotypes: kilBis necessaryforconijugal transfer, whereas kiL4 isresponsible forthesmall-colony morphology on defined media that ischaracteristic ofpKM101-containing strains (the Slophenotype). A numberofplasmic& havebeenfound tocontain genes whoseproducts arepotentially lethal tothebacterial host. TheIncPplasmid RK2 hasthree suchgenes, designated kilA, kilB, andkilC(11). Thelethality ofthese genesis normally inhibited bythree other plasmid-encoded genes, designated korA,korB, andkorC(for kiloverride). korA alone issufficient tocontrol thelethality ofkilA(30). In contrast, korAandkorBtogether arerequired tocontrol the lethality ofkilB (D.H.Bechhofer andD.H.Figurski, manuscript inpreparation), andsimilarly, korAandkorCmust acttogether toprevent thelethal phenotype ofkilC (30). In astrain containing arhomutation, korCalone issufficient to control kilC. Ithasbeenspeculated thatkorApositively effects transcription ofkorC, possibly byantitermination. TheDNA sequence ofthekorAgenehasbeendetermined (1).'- TheF episome hasalsobeenreported tocontain two genes, onehaving thephenotype ofakil geneandtheother having thephenotype ofakorgene(20). Thesegeneshave beenimplicated inenhancing plasmid stability bycoupling hostcell division toplasmid replication. pKM101isa35.4-kilobase (kb)self-transmissible IncN

Stephen C. Winans

Papers

Bacterial evolution by intelligent design.

The Cell-Cell Communication System of Agrobacterium Tumefaciens

Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend.

Cell-Cell Signaling within Crown Gall Tumors

Identification ofpKM101-Encoded LociSpecifying Potentially Lethal GeneProducts