Global regulation of gene expression in response to cysteine availability in Clostridium perfringens.

BACKGROUND: Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail. RESULTS: We first performed a tentative reconstruction of sulfur metabolism in C. perfringens and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, C. perfringens can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (cysK-cysE, cysP1 and cysP2) or an S-box riboswitch (metK and metT). We also showed that the ubiG operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA.In addition, we found that expression of pfoA (theta-toxin), nagL (one of the five genes encoding hyaluronidases) and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the ldh gene encoding the lactate dehydrogenase was induced during cysteine limitation. CONCLUSION: Several key functions for the cellular physiology of this anaerobic bacterium were controlled in response to cysteine availability. While most of the genes involved in sulfur metabolism are regulated by premature termination of transcription, other still uncharacterized mechanisms of regulation participated in the induction of gene expression during cysteine starvation.

Different regulation and roles of lactonases AiiB and AttM in Agrobacterium tumefaciens C58.

The phytopathogen Agrobacterium tumefaciens C58 expresses two lactonases, AttM and AiiB. We showed that expression of the aiiB gene was controlled by agrocinopines A and B and required the agrocinopine-ABC transporter Acc, but was not affected by the level of quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8-HSL). In the presence of agrocinopines, a constructed aiiB mutant accumulated OC8-HSL at a level 10-fold higher than that of the wild-type strain, and showed an exacerbated expression of a key QS-regulated function, conjugation of Ti plasmid (in vitro and in planta), as well as an increase of the number of emerging tumors on the host plant. The expression and acyl-HSL-degrading activity of AttM were evident in the presence of wounded tissues; however, in unwounded plant tumors, the QS-regulated functions were weakly affected in an attM mutant. By contrast, we observed that attM conferred a selective advantage in the course of colonization of plant tumors. Finally, polymerase chain reaction survey of genes attM and aiiB showed that they were not strictly conserved in the genus Agrobacterium. This work proved that the lactonases AttM and AiiB are regulated by different plant signals and are implicated in different functions in the course of the A. tumefaciens C58-host interaction.

Comparative transcriptome analysis of Agrobacterium tumefaciens in response to plant signal salicylic acid, indole-3-acetic acid and gamma-amino butyric acid reveals signalling cross-talk and Agrobacterium--plant co-evolution.

Agrobacterium has evolved sophisticated strategies to perceive and transduce plant-derived cues. Recent studies have found that numerous plant signals, including salicylic acid (SA), indole-3-acetic acid (IAA) and gamma-amino butyric acid (GABA), profoundly affect Agrobacterium-plant interactions. Here we determine and compare the transcriptome profiles of Agrobacterium in response to these three plant signals. Collectively, the transcription of 103, 115 and 95 genes was significantly altered by SA, IAA and GABA respectively. Both distinct cellular responses and overlapping signalling pathways were elicited by these three plant signals. Interestingly, these three plant compounds function additively to shut off the Agrobacterium virulence programme and activate the quorum-quenching machinery. Moreover, the repression of the virulence programme by SA and IAA and the inactivation of quorum-sensing signals by SA and GABA are regulated through independent pathways. Our data indicate that these plant signals, while cross-talk in plant signalling networks, also act as cross-kingdom signals and play redundant roles in tailoring Agrobacterium regulatory pathways, resulting in intensive signalling cross-talk in Agrobacterium. Our results support the notion that Agrobacterium has evolved the ability to hijack plant signals for its own benefit. The complex signalling interplay between Agrobacterium and its plant hosts reflects an exquisite co-evolutionary balance.

Cpe1786/IscR of Clostridium perfringens represses expression of genes involved in Fe-S cluster biogenesis.

Cpe1786 of Clostridium perfringens is an Rrf2-type regulator containing the three-cysteine residues coordinating a Fe-S in IscR, the repressor controlling Fe-S homeostasis in enterobacteria. The cpe1786 gene formed an operon with iscSU involved in Fe-S biogenesis and tmrU. This operon was transcribed from a σA-dependent promoter. We showed that in the heterologous host Bacillus subtilis, Cpe1786, renamed IscRCp, negatively controlled its own transcription. We constructed an iscR mutant in C. perfringens. We then compared the expression profile of strain 13 and of the iscR mutant. IscRCp controlled expression of genes involved in Fe-S biogenesis, in amino acid or sugar metabolisms, in fermentation pathways and in host compound utilization. We then demonstrated, using a ChIP-PCR experiment, that IscRCp interacted with its promoter region in vivo in C. perfringens and with the promoter of cpe2093 encoding an amino acid ABC transporter. We utilized a comparative genomic approach to infer a candidate IscR binding motif and reconstruct IscR regulons in clostridia. We showed that point mutations in the conserved motif of 29 bp identified upstream of iscR decreased the cysteine-dependent repression of iscR mediated by IscRCp.

A fine control of quorum-sensing communication in Agrobacterium tumefaciens.

The bacterial pathogen Agrobacterium tumefaciens produces the quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8HSL) for controlling horizontal transfer of its tumor inducing (Ti) plasmid that carries both the T-DNA and the virulence genes. Over-accumulation of OC8HSL also increases severity of plant symptoms (number of emerging tumors at infection site) by an unknown mechanism. A. tumefaciens strain C58 expresses two lactonases, AttM (BlcC) and AiiB, that cleave OC8HSL and are potential modulators of QS. Recent data highlight the direct contribution of lactonases AttM and AiiB in the control of OC8HSL level and QS-regulated functions such as conjugation of Ti plasmid and seriousness of plant symptoms. Expression of the two lactonases is regulated by different plant signals. A working model of QS in the course of the A. tumefaciens-plant host interaction is proposed and discussed.

GABA controls the level of quorum-sensing signal in Agrobacterium tumefaciens.

The concentration of GABA increases rapidly in wounded plant tissues, but the implication of this GABA pulse for plant-bacteria interactions is not known. Here we reveal that GABA stimulated the inactivation of the N-(3-oxooctanoyl)homoserine lactone (OC8-HSL) quorum-sensing signal (or "quormone") by the Agrobacterium lactonase AttM. GABA induced the expression of the attKLM operon, which was correlated to a decrease in OC8-HSL concentration in Agrobacterium tumefaciens cultures. The Agrobacterium GABA transporter Bra was required for this GABA-signaling pathway. Furthermore, transgenic tobacco plants with elevated GABA levels were less sensitive to A. tumefaciens C58 infection than were wild-type plants. These findings indicate that plant GABA may modulate quorum sensing in A. tumefaciens, thereby affecting its virulence on plants. Whereas GABA is an essential cell-to-cell signal in eukaryotes, here we provide evidence of GABA acting as a signal between eukaryotes and pathogenic bacteria. The GABA signal represents a potential target for the development of a strategy to control the virulence of bacterial pathogens.

Organization and expression of the GSK3/shaggy kinase gene family in the moss Physcomitrella patens suggest early gene multiplication in land plants and an ancestral response to osmotic stress.

GSK3/Shaggy kinases are involved in a wide range of fundamental processes in animal development and metabolism. In angiosperm plants, these kinases are encoded by moderate-sized gene families, which appear to have a complex set of functions. Here, we present the characterization of five members of the GSK3/Shaggy gene family in the bryophyte Physcomitrella patens. The P. patens GSK3/Shaggy kinases (PpSK) are organized in a group of closely related paralogues with respect to their gene sequence and structure. Indeed, a phylogenetic analysis of the GSK3/Shaggy kinase sequences from plants and animals showed that the five PpSK proteins are monophyletic, and closer to subgroups I and IV described in angiosperms. Expression analyses performed by quantitative real-time RT-PCR on a wide range of growing conditions showed that PpSK genes responded only to either desiccation, PEG or sorbitol. As demonstrated by both inductions of marker genes and protonemal cell plasmolyses, these treatments resulted in a hyperosmotic stress. Altogether, these data suggest that (1) GSK3/Shaggy kinase gene multiplication occurred early in plant evolution, before the separation between bryophytes and vascular plants, and (2) both gene loss and duplication occurred in the ancestor of P. patens along with functional gene diversification in angiosperms. However, conservation of the transcriptional responses between Physcomitrella and Arabidopsis suggests the identification of an ancestral response of the GSK3/Shaggy kinases genes to osmotic stress.