Silvia Rüberg

Global Changes in Gene Expression in Sinorhizobium meliloti 1021 under Microoxic and Symbiotic Conditions

Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Sinorhizobium meliloti ExoR and ExoS Proteins Regulate both Succinoglycan and Flagellum Production

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.

Biosynthesis of the exopolysaccharide galactoglucan in Sinorhizobium meliloti is subject to a complex control by the phosphate-dependent regulator PhoB and the proteins ExpG and MucR.

The soil bacterium Sinorhizobium meliloti (Rhizobium meliloti) has the ability to produce the alternative exopolysaccharide galactoglucan (EPS II) in addition to succinoglycan (EPS I). In the wild-type strain EPS II production is induced by phosphate-limiting conditions or by extra copies of the exp gene cluster. Based on similarities to transcriptional regulators of the MarR family, an additional putative regulatory gene, expG, was identified in the exp gene cluster. Using exp-lacZ transcriptional fusions, a stimulating effect of extra copies of this expG gene on the transcription of all exp complementation groups was determined. Phosphate limitation also resulted in increased expression of the exp-lacZ fusions. This increase was reduced in strains characterized by a deletion of expG. The previously reported high level of exp gene transcription in a mucR mutant was further elevated under phosphate-limiting conditions. The expA, expD, expG and expE promoters contain sequences with similarities to the PHO box known as the PhoB-binding site in phosphate-regulated promoters in Escherichia coli. The S. meliloti phoB gene was required for the activation of exp gene expression under phosphate limitation, but not for induction of exp expression by MucR or ExpG.

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: Use of a cosmid and a bacterial artificial chromosome library

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum.

Two New Sinorhizobium meliloti LysR-Type Transcriptional Regulators Required for Nodulation

The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.

A portal for rhizobial genomes: RhizoGATE integrates a Sinorhizobium meliloti genome annotation update with postgenome data

Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti-Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new web-based portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources.

THE REGULATORY PROTEIN MUCR BINDS TO A SHORT DNA REGION LOCATED UPSTREAM OF THE MUCR CODING REGION IN RHIZOBIUM MELILOTI

Abstract The Rhizobium meliloti MucR protein is known to regulate the biosynthesis of the two exopolysaccharides, succinoglycan and galactoglucan. The mucR gene was successfully overexpressed in Escherichia coli BL21 cells by heat shock induction using a two-plasmid system. Cell extracts of the production strain contained about 20% of a polypeptide of 17 kDa apparent molecular mass, corresponding to the size expected for MucR. As shown by an electrophoretic mobility shift assay, these extracts were active in the specific retardation of a 219-bp DNA fragment including 134-bp of the non-coding region upstream of the mucR gene. Primer extension analysis showed that this DNA fragment was located within the transcribed region upstream of the mucR gene. Competition experiments revealed that a 44-bp sequence present within the 134-bp upstream of the mucR gene contained the MucR binding site. Although binding of MucR to this site exhibited a moderate dissociation constant of

Wide-range transcriptional modulating effect of ntrR under microaerobiosis in Sinorhizobium meliloti.

K_{\rm d} \approx 1.4 \times10^{-7}

A homolog of theRhizobium meliloti nitrogen fixation genefixN is involved in the production of a microaerobically induced oxidase activity in the phytopathogenic bacteriumAgrobacterium tumefaciens

M, the reaction was highly specific since fragments containing binding sites for the homologous Ros protein from Agrobacterium tumefaciens were not able to compete for MucR binding.

Isolation of Two New Sinorhizobium meliloti Transcriptional Regulators Required for Nodulation

A mutation in the second gene in the ntrPR operon results in increased expression of nodulation ( nod) and nitrogen fixation ( nif) genes in Sinorhizobium meliloti. Since this pleiotropic effect is particularly pronounced in the presence of external combined nitrogen, a nitrogen regulatory function has been suggested for NtrR. To identify the complete set of protein-coding genes influenced by loss of ntrR function, microarray hybridizations were carried out to compare transcript levels in the wild type and mutant strains grown under aerobic and microaerobic conditions. Of the 6207 genes examined, representing the entire genome of S. meliloti, 7% exhibited altered expression: 4.5% of the genes are affected under oxic, 2.5% under microoxic conditions. 0.4% of all the genes are affected under both oxygen concentrations. A microoxic environment is required for the induction of genes related to symbiotic functions but results in the down-regulation of other (e.g. metabolic) functions. When the alterations in transcription levels at low oxygen concentration in the mutant strain were compared to those of the wild type, a modulating effect of the ntrR mutation was observed. For example, symbiotic nif/fix genes were induced in both strains, but the level of induction was higher in the ntrR mutant. In contrast, genes related to transcription/translation functions were down-regulated in both strains, and the effect was greater in the wild-type strain than in the ntrR mutant. A relatively wide range of functions was affected by this modulating influence, suggesting that ntrR is not a nitrogen regulatory gene. Since genes encoding various unrelated functions were affected, we propose that NtrR may either interfere with general regulatory mechanisms, such as phosphorylation/dephosphorylation, or may influence RNA stability.