The role of sigma factor RpoH1 in the pH stress response of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

Background  

Environmental pH stress constitutes a limiting factor for S. meliloti survival and development. The response to acidic pH stress in S. meliloti is versatile and characterized by the differential expression of genes associated with various cellular functions. The purpose of this study was to gain detailed insight into the participation of sigma factors in the complex stress response system of S. meliloti 1021 using pH stress as an effector.     

Transcriptome analysis of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>during symbiosis

Background  

Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis.     

Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti

Background  

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H₂O₂ and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria.     

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free‐living micro‐oxic and symbiotic conditions

Aim

In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free‐living and symbiotic conditions have been carried out.

Methods and Results

Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD‐dependent oxidase activity when cells were incubated under micro‐oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane‐bound c‐type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb₃ oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N‐free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation.

Conclusions

The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro‐oxic conditions as well as in the expression of the FixO and FixP components of the cbb₃ oxidase present in free‐living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis.

Significance and Impact of the Study

It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb₃ oxidase operating in free‐living micro‐oxic cultures and in bacteroids of E. meliloti have been identified.     

Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti

Background  

S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized.     

Phenotypic and genetic diversity in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">S. medicae</Emphasis> from drought and salt affected regions of Morocco

Background  

Sinorhizobium meliloti and S. medicae are symbiotic nitrogen fixing bacteria in root nodules of forage legume alfalfa (Medicago sativa L.). In Morocco, alfalfa is usually grown in marginal soils of arid and semi-arid regions frequently affected by drought, extremes of temperature and soil pH, soil salinity and heavy metals, which affect biological nitrogen fixing ability of rhizobia and productivity of the host. This study examines phenotypic diversity for tolerance to the above stresses and genotypic diversity at Repetitive Extragenic Pallindromic DNA regions of Sinorhizobium nodulating alfalfa, sampled from marginal soils of arid and semi-arid regions of Morocco.     

The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

Background  

Soil bacteria collectively known as Rhizobium, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a fadD mutant of Sinorhizobium meliloti is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogeniCity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen Xanthomonas campestris, a fadD homolog (rpfB) forms part of a cluster of genes involved in the regulation of pathogeniCity factors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a S. meliloti fadD-linked gene.     

In silico insights into the symbiotic nitrogen fixation in Sinorhizobium meliloti via metabolic reconstruction

Background

Sinorhizobium meliloti is a soil bacterium, known for its capability to establish symbiotic nitrogen fixation (SNF) with leguminous plants such as alfalfa. S. meliloti 1021 is the most extensively studied strain to understand the mechanism of SNF and further to study the legume-microbe interaction. In order to provide insight into the metabolic characteristics underlying the SNF mechanism of S. meliloti 1021, there is an increasing demand to reconstruct a metabolic network for the stage of SNF in S. meliloti 1021.

Results

Through an iterative reconstruction process, a metabolic network during the stage of SNF in S. meliloti 1021 was presented, named as iHZ565, which accounts for 565 genes, 503 internal reactions, and 522 metabolites. Subjected to a novelly defined objective function, the in silico predicted flux distribution was highly consistent with the in vivo evidences reported previously, which proves the robustness of the model. Based on the model, refinement of genome annotation of S. meliloti 1021 was performed and 15 genes were re-annotated properly. There were 19.8% (112) of the 565 metabolic genes included in iHZ565 predicted to be essential for efficient SNF in bacteroids under the in silico microaerobic and nutrient sharing condition.

Conclusions

As the first metabolic network during the stage of SNF in S. meliloti 1021, the manually curated model iHZ565 provides an overview of the major metabolic properties of the SNF bioprocess in S. meliloti 1021. The predicted SNF-required essential genes will facilitate understanding of the key functions in SNF and help identify key genes and design experiments for further validation. The model iHZ565 can be used as a knowledge-based framework for better understanding the symbiotic relationship between rhizobia and legumes, ultimately, uncovering the mechanism of nitrogen fixation in bacteroids and providing new strategies to efficiently improve biological nitrogen fixation.     

Genome-Wide Identification and Expression Profiling Analysis of the Aux/IAA Gene Family in Medicago truncatula during the Early Phase of Sinorhizobium meliloti Infection

Background

Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation.

Principal Findings

Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application.

Conclusion

The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.     

Auxin and nitric oxide control indeterminate nodule formation

Background  

Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using Sinorhizobium meliloti and Rhizobium leguminosarum bacteria genetically engineered for increased auxin synthesis.     

Rapid acid treatment of <Emphasis Type="Italic">Escherichia coli</Emphasis>: transcriptomic response and recovery

Background  

Many E. coli genes show pH-dependent expression during logarithmic growth in acid (pH 5–6) or in base (pH 8–9). The effect of rapid pH change, however, has rarely been tested. Rapid acid treatment could distinguish between genes responding to external pH, and genes responding to cytoplasmic acidification, which occurs transiently following rapid external acidification. It could reveal previously unknown acid-stress genes whose effects are transient, as well as show which acid-stress genes have a delayed response.     

Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition inEscherichia coli K-12

Background  

InEscherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated.     

A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti

Background  

Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown.     

A metabolomic approach to characterize the acid-tolerance response in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

Introduction

Sinorhizobium meliloti establishes a symbiosis with Medicago species where the bacterium fixes atmospheric nitrogen for plant nutrition. To achieve a successful symbiosis, however, both partners need to withstand biotic and abiotic stresses within the soil, especially that of excess acid, to which the Medicago-Sinorhizobium symbiotic system is widely recognized as being highly sensitive.

Objective

To cope with low pH, S. meliloti can undergo an acid-tolerance response (ATR(+)) that not only enables a better survival but also constitutes a more competitive phenotype for Medicago sativa nodulation under acid and neutral conditions. To characterize this phenotype, we employed metabolomics to investigate the biochemical changes operating in ATR(+) cells.

Methods

A gas chromatography/mass spectrometry approach was used on S. meliloti 2011 cultures showing ATR(+) and ATR(?) phenotypes. After an univariate and multivariate statistical analysis, enzymatic activities and/or reserve carbohydrates characterizing ATR(+) phenotypes were determined.

Results

Two distinctive populations were clearly defined in cultures grown in acid and neutral pH based on the metabolites present. A shift occurred in the carbon-catabolic pathways, potentially supplying NAD(P)H equivalents for use in other metabolic reactions and/or for maintaining intracellular-pH homeostasis. Furthermore, among the mechanisms related to acid resistance, the ATR(+) phenotype was also characterized by lactate production, envelope modification, and carbon-overflow metabolism.

Conclusions

Acid-challenged S. meliloti exhibited several changes in different metabolic pathways that, in specific instances, could be identified and related to responses observed in other bacteria under various abiotic stresses. Some of the observed changes included modifications in the pentose-phosphate pathway (PPP), the exopolysaccharide biosynthesis, and in the myo-inositol degradation intermediates. Such modifications are part of a metabolic adaptation in the rhizobia that, as previously reported, is associated to improved phenotypes of acid tolerance and nodulation competitiveness.

     

Expression of novel carotenoid biosynthesis genes from Enterococcus gilvus improves the multistress tolerance of Lactococcus lactis

Aims

To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid‐producing engineered MG1363 strain under stress condition was investigated.

Methods and Results

We cloned carotenoid biosynthesis genes from yellow‐pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid‐producing L. lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C₃₀ carotenoid 4,4′‐diaponeurosporene. After exposure to 32 mmol l^?1 H₂O₂, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml^?1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7‐, 6.8‐, 8.8‐ and 4.4‐fold higher, respectively, than those of MG1363 strain harbouring the empty vector pRH100.

Conclusions

The expression of carotenoid biosynthesis genes from Ent. gilvus improves the multistress tolerance of L. lactis.

Significance and Impact of the study

First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production.     

Phylogenetic and regulatory region analysis of <Emphasis Type="Italic">Wnt5</Emphasis> genes reveals conservation of a regulatory module with putative implication in pancreas development

Background  

Wnt5 genes belong to the large Wnt family, encoding proteins implicated into several tumorigenic and developmental processes. Phylogenetic analyses showed that Wnt5 gene has been duplicated at the divergence time of gnathostomata from agnatha. Interestingly, experimental data for some species indicated that only one of the two Wnt5 paralogs participates in the development of the endocrine pancreas. The purpose of this paper is to reexamine the phylogenetic history of the Wnt5 developmental regulators and investigate the functional shift between paralogs through comparative genomics.     

Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

Background  

PhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.     

Responses of community structure of amoA‐encoding archaea and ammonia‐oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate

Aims

To investigate community shifts of amoA‐encoding archaea (AEA) and ammonia‐oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process.

Methods and Results

A laboratory‐scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49–63 NH₃ g m^?3 day^?1. The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real‐time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively.

Conclusions

The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH.

Significance and Impact of the Study

One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.