B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase. Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine. The B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control B activity in response to environmental stress. However, none of these is sufficient to explain the enhanced stationary-phase activation of B in response to glucose. We show here that the four genes previously identified in the sigB operon constitute the downstream half of an eight-gene operon. The complete sigB operon is preceded by a A -like promoter (P A ) and has the order P A -orfR-orfSorfT-orfU-P B -rsbV-rsbW-sigB-rsbX, where rsb stands for regulator of sigma-B and the previously identified B -dependent promoter (P B ) is an internal promoter preceding the downstream four-gene cluster. Although the genes downstream of P B were also transcribed by polymerase activity originating at P A , this transcription into the downstream cluster was not essential for normal induction of a B -dependent ctc-lacZ fusion. However, deletion of all four upstream open reading frames was found to interfere with induction of the ctc-lacZ fusion in response to glucose. Additional deletion analysis and complementation studies showed that orfU was required for full glucose induction of B -dependent genes. orfU encodes a trans-acting, positive factor with significant sequence identity to the RsbX negative regulator of B . On the basis of these results, we rename orfU as rsbU to symbolize the regulatory role of its product.The purpose of many prokaryotic signal transduction pathways is to change the level of transcription of a set of target genes in response to a particular environmental signal. Stress and starvation signals elicit particularly dramatic changes in bacterial gene expression (15,19,21,27,35,46). In a number of cases, these stress and starvation signals have been shown to activate alternative factors that associate with RNA polymerase and reprogram the promoter recognition specificity of the enzyme, thus allowing the expression of new sets of target genes (18,19,32,48). Because factors are such powerful regulators of bacterial gene expression, the signal transduction pathways that control their activation are an area of vigorous investigation.The alternative transcription factor B of Bacillus subtilis is activated upon entry into the stationary growth phase, and this stationary-phase activation is substantially enhanced when cells are grown in a rich medium containing high levels of glucose and glutamine (7,9,25). Significantly, recent work has shown that B is also activated in logarithmically growing cells by a variety of environmental stresses that limit growth, including hyperosmosis, heat, oxidative stress, and ethanol shock (4, 6, 51). These finding...

Agrobacterium tumefaciens is capable of transferring and integrating an oncogenic T-DNA (transferred DNA) from its tumor-inducing (Ti) plasmid into dicotyledonous plants. This transfer requires that the virulence genes (vir regulon) be induced by plant signals such as acetosyringone in an acidic environment. Salicylic acid (SA) is a key signal molecule in regulating plant defense against pathogens. However, how SA influences Agrobacterium and its interactions with plants is poorly understood. Here we show that SA can directly shut down the expression of the vir regulon. SA specifically inhibited the expression of the Agrobacterium virA/G two-component regulatory system that tightly controls the expression of the vir regulon including the repABC operon on the Ti plasmid. We provide evidence suggesting that SA attenuates the function of the VirA kinase domain. Independent of its effect on the vir regulon, SA up-regulated the attKLM operon, which functions in degrading the bacterial quormone N-acylhomoserine lactone. Plants defective in SA accumulation were more susceptible to Agrobacterium infection, whereas plants overproducing SA were relatively recalcitrant to tumor formation. Our results illustrate that SA, besides its well known function in regulating plant defense, can also interfere directly with several aspects of the Agrobacterium infection process.two-component system ͉ tumorigenesis ͉ defense response ͉ rhizosphere ͉ plant-microbe interaction

The genetic manipulation of Agrobacterium tumefaciens is used to facilitate studies of bacterial gene functions or as a first step in introducing genetic material into transformable plant cells through the use of T-DNA binary vectors. Three methods are commonly used. Transformation with purified plasmid can be done with either electroporation or a simple freeze/thaw transformation method. Alternatively, a mobilizable plasmid can be placed into Agrobacterium using the triparental mating method. Here we present three detailed protocols for Agrobacterium strain construction using electroporation, the freeze/thaw method of transformation, and triparental mating.

proP of Escherichia coli encodes an active transport system for proline and glycine betaine which is activated by both hyperosmolarity and amino acid-limited growth. proP DNA sequences far upstream from the translational start site are strongly homologous to the promoter of proU, an operon that specifies another osmoregulated glycine betaine transport system. Mutation and deletion analysis of proP and primer extension experiments established that this promoter, P1, was responsible for proP's strong expression in minimal medium and its response to osmotic signals. When cells were grown in complex medium, expression from a proP-lacZ fusion was induced three-to fourfold as growth slowed and cells entered stationary phase. Stationary-phase induction was dependent on rpoS, which encodes a stationary-phase sigma factor. Deletion of 158 bp of the untranslated leader sequence between P1 and the proP structural gene abolished rpoS-dependent stationary-phase regulation. Transcription initiation detected by primer extension within this region was absent in an rpoS mutant. proP is therefore a member of the growing class of S -dependent genes which respond to both stationary-phase and hyperosmolarity signals.The growth rate of Escherichia coli in hyperosmotic media is enhanced by the accumulation of a limited number of organic solutes. The preferred solute is glycine betaine (10, 38), which is accumulated by two transport systems, proP and proU. proP encodes an integral membrane protein (7) which transports glycine betaine and proline with low affinity (2, 4, 28). ProP regulation occurs at the level of transcription and through control of transport activity. proP-lacZ transcriptional fusions show a two-to fivefold increase in activity when cells are osmotically stressed (11,31). Transport of glycine betaine and proline by ProP is also increased by osmotic upshock (14, 28) and amino acid limitation (14,37).proU encodes a high-affinity glycine betaine uptake system (5, 8, 28). Like ProP, ProU is regulated at the level of transcription and through control of transport activity. Under hyperosmotic conditions, the activity of proU-lacZ operon fusions increases 60-to 100-fold (3,5,29), and transport of glycine betaine by the proU system increases (28). Unlike proP, proU transcription is not induced in stationary phase, although several other osmotically responsive genes, e.g., osmY, osmB, otsB, and otsA, respond to growth phase signals (15,18,40).Certain observations have suggested that proP and proU may be regulated in a similar manner. First, induction from proPlacZ and proU-lacZ operon fusions is rapid upon osmotic shock (16). Second, the proP gene sequence was recently reported (7), and a region 190 bp upstream of the translation initiation site is strongly homologous to the promoter of proU. In this investigation, we studied the regulation of proP, asking whether that putative promoter functions in transcription initiation. We found that this promoter was responsible for strong basal expression and response to osmotic signals...

The genetic systems of bacteria that have the ability to use organic pollutants as carbon and energy sources can be adapted to create bacterial biosensors for the detection of industrial pollution. The creation of bacterial biosensors is hampered by a lack of information about the genetic systems that control production of bacterial enzymes that metabolize pollutants. We have attempted to overcome this problem through modification of DmpR, a regulatory protein for the phenol degradation pathway of Pseudomonas sp. strain CF600. The phenol detection capacity of DmpR was altered by using mutagenic PCR targeted to the DmpR sensor domain. DmpR mutants were identified that both increased sensitivity to the phenolic effectors of wild-type DmpR and increased the range of molecules detected. The phenol detection characteristics of seven DmpR mutants were demonstrated through their ability to activate transcription of a lacZ reporter gene. Effectors of the DmpR derivatives included phenol, 2-chlorophenol, 2,4-dichlorophenol, 4-chloro-3-methylphenol, 2,4-dimethylphenol, 2-nitrophenol, and 4-nitrophenol.