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.

The HPrK kinase/phosphatase is a common component of the phosphotransferase system (PTS) of grampositive bacteria and regulates catabolite repression through phosphorylation/dephosphorylation of its substrate, the PTS protein HPr, at a conserved serine residue. Phosphorylation of HPr by HPrK also affects additional phosphorylation of HPr by the PTS enzyme EI at a conserved histidine residue. Sinorhizobium meliloti can live as symbionts inside legume root nodules or as free-living organisms and is one of the relatively rare gram-negative bacteria known to have a gene encoding HPrK. We have constructed S. meliloti mutants that lack HPrK or that lack key amino acids in HPr that are likely phosphorylated by HPrK and EI. Deletion of hprK in S. meliloti enhanced catabolite repression caused by succinate, as did an S53A substitution in HPr. Introduction of an H22A substitution into HPr alleviated the strong catabolite repression phenotypes of strains carrying ⌬hprK or hpr(S53A) mutations, demonstrating that HPr-His22-P is needed for strong catabolite repression. Furthermore, strains with a hpr(H22A) allele exhibited relaxed catabolite repression. These results suggest that HPrK phosphorylates HPr at the serine-53 residue, that HPr-Ser53-P inhibits phosphorylation at the histidine-22 residue, and that HPr-His22-P enhances catabolite repression in the presence of succinate. Additional experiments show that ⌬hprK mutants overproduce exopolysaccharides and form nodules that do not fix nitrogen.

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...

During the course of the development of nitrogen-fixing root nodules induced by Sinorhizobium meliloti on the model plant Medicago truncatula, tubules called infection threads are cooperatively constructed to deliver the bacterial symbiont from the root surface to cells in the interior of the root and developing nodule. Three-dimensional reconstructions of infection threads inside M. truncatula nodules showed that the threads formed relatively simple, tree-like networks. Some characteristics of thread networks, such as branch length, branch density, and branch surface-to-volume ratios, were remarkably constant across nodules in different stages of development. The overall direction of growth of the networks changed as nodules developed. In 5-d-old nodules, the overall growth of the network was directed inward toward the root. However, well-defined regions of these young networks displayed an outward growth bias, indicating that they were likely in the process of repolarizing their direction of development in response to the formation of the outward-growing nodule meristem. In 10-and 30-d-old nodules, the branches of the network grew outward toward the meristem and away from the roots on which the nodules developed.

SummaryThe need for clean water has risen exponentially over the globe. Millions of people are affected daily by a lack of clean water, especially women and children, as much of their day is dedicated to collecting water. The global water crisis not only has severe medical implications, but social, political, and economic consequences as well. The Institute of Catholic Bioethics at Saint Joseph’s University has recognized this, and has designed a slow-sand water filter that is accessible, cost-effective, and sustainable. Through the implementation of the Institute’s slow-sand water filter and the utilization of microfinancing services, developing countries will not only have access to clean, drinkable water, but will also have the opportunity to break out of a devastating cycle of poverty.

Summary Linking microbial activity with ecosystem function is a continuing goal among ecologists focusing their efforts below ground in terrestrial ecosystems. Genomic approaches, using DNA and RNA extracted from soil to characterize types of microbes present and genes expressed in soil, are promising, but, the required destructive harvest confounds spatial and temporal information. Microbiosensors offer a gene‐based way to examine microbial perception of, and response to, the soil environment non‐destructively, with high spatial and temporal resolution. In this mini‐review, we explore the promise, challenges and tradeoffs associated with the design and deployment of microbiosensors in soil, as well as the interpretation of information derived from them ‘live from the soil grain’. Both the promises and challenges are caused by the facts that: micro‐biosensors are living organisms with specific traits; they come from a phylogenetically diverse but restricted subsample of enormous soil microbial diversity; they are responsive to internal and external influences on multiple cellular processes (not just promoter induction); and they become part of food chains in non‐sterile soils. We examine each of these characteristics, and associated blessings and curses, in the context of using microbiosensors to explore microbial soil ecology.

The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 M CdCl 2 ), permitting long-term community monitoring. The broad-host-range IncP␣ plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd r or Ni r density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc-and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni r transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.

The effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the TOL plasmid between Pseudomonas spp. was investigated by examinations of filter matings. A Pseudomonas putida KT2442-derived strain carrying a gfp-tagged variant of the TOL plasmid was used as a donor, and both restriction-deficient (PAO1162N) and -proficient (PAO2002N) Pseudomonas aeruginosa strains were used as recipients. The in situ enumeration of conjugation events allowed us to obtain frequency estimates that were unbiased by transconjugant growth or plasmid retransfer. We observed a strong dependence of the plasmid transfer frequency on the initial donor-to-recipient ratio of surface matings, which invalidated the use of mass action-based plasmid transfer kinetic estimators. Careful control of the initial parental cell densities permitted evaluations of the true effects of restriction proficiency and toxicant exposure on TOL transfer. At standard donor-to-recipient ratios (10 ؊3 for PAO1162N and 2 ؋ 10 1 for PAO2002N) and total cell densities (10 5 cells/mm 2 for PAO1162N and 10 6 cells/mm 2 for PAO2002N), plasmid transfer frequencies without toxicant exposure were approximately 10 ؊7 (events/ mm 2 ) ؊1 for PAO1162N and 10 ؊11 (events/mm 2 ) ؊1 for PAO2002N based on in situ observations of conjugation events. The enumeration of transconjugants via selective plating yielded transfer frequencies that were up to 1 order of magnitude lower. Premating exposure to sodium dodecyl sulfate (1 to 10 mM) significantly increased the transfer frequency for the restriction-proficient strain PAO2002N (P < 0.05) but not for the restriction-deficient strain PAO1162N. On the other hand, premating exposure to ethanol, toluene, or phenol had no positive effect on the plasmid transfer frequency. Clearly, restriction proficiency provides a strong barrier to interspecific transfer of the TOL plasmid, and this barrier was only marginally attenuated by recipient exposure to toxicants within the ranges examined.Plasmid transfer is considered an important factor in the adaptation of microbial communities to environmental changes and in bacterial evolution (16). The transfer of plasmids has been demonstrated in situations in which it confers a selective advantage (e.g., the transfer of plasmids that carry catabolic genes in pollutant-laden environments [4,14,19] and of plasmids that carry metal resistance genes in metal-stressed environments [33]) but also when there is apparently no selective advantage (20, 23). The kinetics of conjugal plasmid transfer are influenced by many factors, including the type of organisms involved, the physiological state of the donor (18, 28), energy or nutrient availability (21, 27), and bacterial distribution and density (2, 20, 30), among others. If plasmid transfer is indeed part of an adaptive response to stress, then stress should increase the ability of donors to transfer or of recipients to acquire a plasmid. For identification of the direct effects of toxicant stress on plasmid transfer rates,...