Elizaveta Krol

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.

Global transcriptional analysis of the phosphate starvation response in Sinorhizobium meliloti strains 1021 and 2011

The global response to phosphate starvation was analysed at the transcriptional level in two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011. The Pho regulon is known to be induced by PhoB under conditions of phosphate limitation. Ninety-eight genes were found to be significantly induced (more than three-fold) in a phoB -dependent manner in phosphate-stressed cells, and phoB -independent repression of 86 genes was observed. Possible roles of these genes in the phosphate stress response are discussed. Twenty new putative PHO box sequences were identified in regions upstream of 17 of the transcriptional units that showed phoB -dependent, or partially phoB -dependent, regulation, indicating direct regulation of these genes by PhoB. Despite the overall similarity between the phosphate stress responses in Rm1021 and Rm2011, lower induction rates were found for a set of phoB -dependent genes in Rm1021. Moreover, Rm1021 exhibited moderate constitutive activation of 12 phosphate starvation-inducible, phoB -dependent genes when cells were grown in a complex medium. A 1-bp deletion was observed in the pstC ORF in Rm1021, which results in truncation of the protein product. This mutation is probably responsible for the expression of phosphate starvation-inducible genes in Rm1021 in the absence of phosphate stress.

Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021.

BackgroundSinorhizobium meliloti is a soil-dwelling α-proteobacterium that possesses a large, tripartite genome and engages in a nitrogen fixing symbiosis with its plant hosts. Although much is known about this important model organism, global characterization of genetic regulatory circuits has been hampered by a lack of information about transcription and promoters.ResultsUsing an RNAseq approach and RNA populations representing 16 different growth and stress conditions, we comprehensively mapped S. meliloti transcription start sites (TSS). Our work identified 17,001 TSS that we grouped into six categories based on the genomic context of their transcripts: mRNA (4,430 TSS assigned to 2,657 protein-coding genes), leaderless mRNAs (171), putative mRNAs (425), internal sense transcripts (7,650), antisense RNA (3,720), and trans-encoded sRNAs (605). We used this TSS information to identify transcription factor binding sites and putative promoter sequences recognized by seven of the 15 known S. meliloti σ factors σ70, σ54, σH1, σH2, σE1, σE2, and σE9). Altogether, we predicted 2,770 new promoter sequences, including 1,302 located upstream of protein coding genes and 722 located upstream of antisense RNA or trans-encoded sRNA genes. To validate promoter predictions for targets of the general stress response σ factor, RpoE2 (σE2), we identified rpoE2-dependent genes using microarrays and confirmed TSS for a subset of these by 5′ RACE mapping.ConclusionsBy identifying TSS and promoters on a global scale, our work provides a firm foundation for the continued study of S. meliloti gene expression with relation to gene organization, σ factors and other transcription factors, and regulatory RNAs.

Sinorhizobium meliloti regulator MucR couples exopolysaccharide synthesis and motility.

In order to enter symbiosis with its legume partner, Sinorhizobium meliloti requires regulatory systems for the appropriate responses to its environment. For example, motility is required for the chemotactic movement of bacteria toward the compounds released by its host, and exopolysaccharides (EPS) are required for bacterial attachment to the root or for invasion of the infection thread. Previous research has shown that ExoR/ExoS/ChvI as well as the ExpR/Sin quorum-sensing system inversely regulate both motility and EPS production, although the regulation mechanisms were unknown. We were able to attribute the ExpR-mediated regulation of motility to the ability of ExpR to bind a DNA sequence upstream of visN when activated by N-acyl-homoserine lactone. Furthermore, MucR, previously characterized as a regulator of EPS production, also affected motility. MucR inhibited expression of rem encoding an activator of motility gene expression and, consequently, the expression of Rem-regulated genes such as flaF and flgG. Binding of MucR to the rem promoter region was demonstrated and a sequence motif similar to the previously identified MucR binding consensus was identified within this region. The swarming ability of S. meliloti Rm2011 was shown to depend on a functional ExpR/Sin quorum-sensing system and the production of both flagella and EPS. Finally, we propose a model for the coordination of motility and EPS synthesis in S. meliloti.

Competitive and Cooperative Effects in Quorum-Sensing-Regulated Galactoglucan Biosynthesis in Sinorhizobium meliloti

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti possesses the Sin quorum-sensing system based on N-acyl homoserine lactones (AHLs) as signal molecules. The Sin system consists of SinI, the AHL synthase, and SinR, the LuxR-type regulator. This system regulates the expression of a multitude of S. meliloti genes through ExpR, another LuxR-type regulator. Analysis of the activity of the sinI promoter showed that the expression of sinI is dependent on sinR and enhanced by a combination of expR and Sin AHLs. The characterization of the ExpR binding site upstream of sinI and the identification of binding sites upstream of the galactoglucan biosynthesis genes wgaA (expA1) and wgeA (expE1) allowed the definition of a consensus sequence for these binding sites. Based on this consensus, two additional ExpR binding sites in the promoter regions of exoI and exsH, two genes related to the production of succinoglycan, were found. The specific binding of ExpR to the wgaA and wgeA promoters was enhanced in the presence of oxo-C(14)-HL. Positive regulation of the galactoglucan biosynthesis genes by ExpR was shown to be dependent on WggR (ExpG) and influenced by MucR, both of which are previously characterized regulators of these genes. Based on these results, a reworked model of the Sin-ExpR quorum-sensing regulation scheme of galactoglucan production in S. meliloti is suggested.

ppGpp in Sinorhizobium meliloti: biosynthesis in response to sudden nutritional downshifts and modulation of the transcriptome

Sinorhizobium meliloti Rm2011 responds to sudden shifts to nitrogen or carbon starvation conditions by an accumulation of the stringent response alarmone ppGpp and remodelling of the transcriptome. The gene product of relA, RelSm, responsible for synthesis of ppGpp, shows functional similarities to E. coli SpoT. Using promoter–egfp gene fusions, we showed that in Rm2011 relA is expressed at a low rate, as a readthrough from the rpoZ promoter and from its own weak promoter. The low level of relA expression is physiologically relevant, since overexpression of RelSm inhibits ppGpp accumulation. The N‐terminal portion of RelSm is required for ppGpp degradation in nutrient‐sufficient cells and might be involved in regulation of the ppGpp synthase and hydrolase activities of the protein. Expression profiling of S. meliloti subjected to sudden nitrogen or carbon downshifts revealed that repression of ‘house‐keeping’ genes is largely dependent on relA whereas activation of gene targets of the stress sigma factor RpoE2 occurred independently of relA. The regulatory genes nifA, ntrB, aniA and sinR, as well as genes related to modulation of protein biosynthesis and nucleotide catabolism, were induced in a relA‐dependent manner. dksA was required for the majority of the relA‐dependent regulations.

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.

Rhizobial homologs of the fatty acid transporter FadL facilitate perception of long-chain acyl-homoserine lactone signals

Significance Bacterial intercellular communication is crucial for developing population and community structures and for pathogenic and symbiotic interactions with eukaryotic hosts. Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) in quorum sensing-related signaling. Although it is likely that specific transport mechanisms are required for long-chain AHLs to overcome the outer membrane, mechanisms promoting uptake have not been reported so far. Here we present evidence that homologs of the outer membrane long-chain fatty acid transporter FadL facilitate uptake of long-chain AHLs, which closes an important gap in our understanding of quorum sensing signaling. Our findings suggest that bacteria responding to long-chain AHLs have evolved specificity of FadL toward these signal molecules and have partly lost the ability to transport long-chain fatty acid by this protein. Quorum sensing (QS) using N-acyl homoserine lactones (AHLs) as signal molecules is a common strategy used by diverse Gram-negative bacteria. A widespread mechanism of AHL sensing involves binding of these molecules by cytosolic LuxR-type transcriptional regulators, which requires uptake of external AHLs. The outer membrane is supposed to be an efficient barrier for diffusion of long-chain AHLs. Here we report evidence that in Sinorhizobium meliloti, sensing of AHLs with acyl chains composed of 14 or more carbons is facilitated by the outer membrane protein FadLSm, a homolog of the Escherichia coli FadLEc long-chain fatty acid transporter. The effect of fadLSm on AHL sensing was more prominent for longer and more hydrophobic signal molecules. Using reporter gene fusions to QS target genes, we found that fadLSm increased AHL sensitivity and accelerated the course of QS. In contrast to FadLEc, FadLSm did not support uptake of oleic acid, but did contribute to growth on palmitoleic acid. FadLSm homologs from related symbiotic α-rhizobia and the plant pathogen Agrobacterium tumefaciens differed in their ability to facilitate long-chain AHL sensing or to support growth on oleic acid. FadLAt was found to be ineffective toward long-chain AHLs. We obtained evidence that the predicted extracellular loop 5 of FadLSm and further α-rhizobial FadL proteins contains determinants of specificity to long-chain AHLs. Replacement of a part of loop 5 by the corresponding region from α-rhizobial FadL proteins transferred sensitivity for long-chain AHLs to FadLAt.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

Significance Cyclic dimeric GMP (c-di-GMP) has emerged as ubiquitous bacterial second messenger, regulating multiple cellular functions, such as cell cycle, virulence, and biofilm formation. However, our knowledge on the molecular inventory, diversity, and function of c-di-GMP receptors, and the molecular evolution of c-di-GMP–responsive proteins is still incomplete. We have identified a class of c-di-GMP–responsive transcription factors, strikingly illustrating how a classical transcription factor has acquired the ability to sense this signaling molecule. The mode of c-di-GMP binding to the AraC-like transcription factor CuxR is highly reminiscent to that of the PilZ domain, the prototypic c-di-GMP receptor. PilZ and CuxR provide an example of convergent evolution in which c-di-GMP binding sites of similar topology have evolved independently in two distinct protein families. Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal “RxxxR” motif and a β-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP–responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP–responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.