Identifying the types and distributions of organic substrates that support microbial activities around plant roots is essential for a full understanding of plant-microbe interactions and rhizosphere ecology. We have constructed a strain of the soil bacterium Sinorhizobium meliloti containing a gfp gene fused to the melA promoter which is induced on exposure to galactose and galactosides. We used the fusion strain as a biosensor to determine that galactosides are released from the seeds of several different legume species during germination and are also released from roots of alfalfa seedlings growing on artificial medium. Galactoside presence in seed wash and sterile root washes was confirmed by HPLC. Experiments examining microbial growth on ␣-galactosides in seed wash suggested that ␣-galactoside utilization could play an important role in supporting growth of S. meliloti near germinating seeds of alfalfa. When inoculated into microcosms containing legumes or grasses, the biosensor allowed us to visualize the localized presence of galactosides on and around roots in unsterilized soil, as well as the grazing of fluorescent bacteria by protozoa. Galactosides were present in patches around zones of lateral root initiation and around roots hairs, but not around root tips. Such biosensors can reveal intriguing aspects of the environment and the physiology of the free-living soil S. meliloti before and during the establishment of nodulation, and they provide a nondestructive, spatially explicit method for examining rhizosphere soil chemical composition.Rhizobium ͉ GFP ͉ protozoa ͉ sugars

Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIA Man -type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of ␣-and ␤-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecularweight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIA Man ) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.

When they are available, Sinorhizobium meliloti utilizes C 4 -dicarboxylic acids as preferred carbon sources for growth while suppressing the utilization of some secondary carbon sources such as ␣-and ␤-galactosides. The phenomenon of using succinate as the sole carbon source in the presence of secondary carbon sources is termed succinate-mediated catabolite repression (SMCR). Genetic screening identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown in succinate plus lactose, maltose, or raffinose. sma0113 and the gene immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of catabolite repression compared to the wild type. sma0114 in-frame deletion mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires sma0113. Sma0113 may use its five PAS domains for redox level or energy state monitoring and use that information to regulate catabolite repression and related responses.Sinorhizobium, Rhizobium, Bradyrhizobium, and Azorhizobium (rhizobia) are important nitrogen-fixing prokaryotes. These grow in the soil as free-living organisms but can also live as nitrogen-fixing symbionts inside roots of plants belonging to the family Leguminosae (8,11,21,33,41,54). Rhizobia are able to utilize a wide range of compounds as carbon sources, such as sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates. Studies have shown that the C 4 -dicarboxylic TCA cycle intermediates succinate, fumarate, and malate support high rates of growth in laboratory medium and are used by rhizobia in preference to carbon sources including glucose, fructose, galactose, lactose, and myo-inositol (23,26,44,63).The phenomenon of Sinorhizobium meliloti utilizing succinate and similar C 4 -dicarboxylic acids in preference to other compounds is called succinate-mediated catabolite repression (SMCR) (9). One of the first reports of catabolite repression in S. meliloti (then Rhizobium meliloti) showed that S. meliloti exhibited diauxic growth in a medium containing 0.2% succinate and 0.2% lactose as carbon sources (63). This study also showed that the production of ␤-galactosidase was repressed when succinate and lactose were present together and that it increased to higher levels after succinate had been exhausted from the medium.Succinate and other C 4 -dicarboxylic acids are sensed and transported by the Dct (dicarboxylate transport) system which is encoded by dctA, dctB, and dctD (48,66,(69)(70)(71). DctB is activated by the presence of C 4 -dicarboxylic acids and autophosphorylates. Activated DctB phosphorylates DctD, which then binds upstream of dctA along with 54 -RNA polymerase to initiate transcription (70). dctA encodes the permease required for transport of succinate and other C 4 -dicarboxylic acids. When succinate is in abundance, S. meliloti will preferentially import this carbon source for metabolism and in...

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the ␣-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other ␣-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of ␤-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of ␣-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce ␤-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinatemediated catabolite repression. Thus, some raffinose could be transported by the overproduced ␤-galactoside transporters and cause the induction of ␣-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a ␤-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.Bacteria belonging to the genera Sinorhizobium, Rhizobium, and Bradyrhizobium are members of the ␣-proteobacteria, a fascinating group of organisms, many of which are intracellular symbionts or pathogens. Sinorhizobium meliloti can grow in soil as free-living organisms but can also live as nitrogen-fixing symbionts inside root nodules of alfalfa and a few other plants belonging to the family Leguminosae (3,9,15,19,22,34).Free-living S. meliloti, like many heterotrophic bacteria, utilizes a wide variety of compounds as sources of carbon for growth. S. meliloti can utilize ␣-galactosides in laboratory medium and also when growing in the rhizospheres of host and nonhost plants (4). The utilization of ␣-galactosides requires genes which are part of an operon located on pSymB, a 1.7-Mb plasmid, in S. meliloti (Fig. 1) (7, 12). The agpA gene encodes a 77-kDa periplasmic protein that is required for ␣-galactoside transport and that is similar to periplasmic binding protein components of the oligopeptide family of permeases ( Fig. 1) (12). ...

This lab reported previously that the plating efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G 1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak plating efficiency occurred as cells cycled out of G 1 and into S phase, suggesting that the enhanced plating efficiency was due to cellular activities present in late G 1 /early S phase. We have found, however, that the enhanced plating efficiency did not occur when cells were synchronized by alternative methods. We now report that the plating efficiency of ICP0 ؊ viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the plating and replication efficiencies of ICP0 ؊ viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced plating efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0 ؊ virus plating efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0 ؊ viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.