Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria.

RNA helicases-central enzymes in RNA metabolism-often feature intrinsically disordered regions (IDRs) that enable phase separation and complex molecular interactions. In the bacterial pathogen Pseudomonas aeruginosa, the non-redundant RhlE1 and RhlE2 RNA helicases share a conserved REC catalytic core but differ in C-terminal IDRs. Here, we show how the IDR diversity defines RhlE RNA helicase specificity of function. Both IDRs facilitate RNA binding and phase separation, localizing proteins in cytoplasmic clusters. However, RhlE2 IDR is more efficient in enhancing REC core RNA unwinding, exhibits a greater tendency for phase separation, and interacts with the RNase E endonuclease, a crucial player in mRNA degradation. Swapping IDRs results in chimeric proteins that are biochemically active but functionally distinct as compared to their native counterparts. The RECRhlE1-IDRRhlE2 chimera improves cold growth of a rhlE1 mutant, gains interaction with RNase E and affects a subset of both RhlE1 and RhlE2 RNA targets. The RECRhlE2-IDRRhlE1 chimera instead hampers bacterial growth at low temperatures in the absence of RhlE1, with its detrimental effect linked to aberrant RNA droplets. By showing that IDRs modulate both protein core activities and subcellular localization, our study defines the impact of IDR diversity on the functional differentiation of RNA helicases.

Multiple Roles of c-di-GMP Signaling in Bacterial Pathogenesis.

The intracellular signaling molecule cyclic di-GMP (c-di-GMP) regulates the lifestyle of bacteria and controls many key functions and mechanisms. In the case of bacterial pathogens, a wide variety of virulence lifestyle factors have been shown to be regulated by c-di-GMP. Evidence of the importance of this molecule for bacterial pathogenesis has become so great that new antimicrobial agents are tested for their capacity of targeting c-di-GMP signaling. This review summarizes the current knowledge on this topic and reveals its application for the development of new antivirulence intervention strategies.

KaiC-like proteins contribute to stress resistance and biofilm formation in environmental Pseudomonas species.

KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologues of this protein are widespread among nonphotosynthetic bacteria, but the function, interaction network, and mechanism of action of these proteins are still largely unknown. Here, we focus on KaiC homologues found in environmental Pseudomonas species. Using bioinformatics, we describe the distribution of this protein family in the genus and reveal a conserved interaction network comprising a histidine kinase and response regulator. We characterize experimentally the only KaiC homologue present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in biofilm production in both species. KaiC homologues are found in different phosphorylation states and physically interact with a cognate histidine kinase and response regulator. In contrast with cyanobacterial counterparts, the expression and phosphorylation of KaiC homologues do not correlate with light variations under 12:12 light: dark cycles in either Pseudomonas species, and KaiC itself is not required to support a light-driven behaviour in P. putida. Overall, this suggests that KaiC homologues in Pseudomonas species are involved in environmental stress resistance but not in responses to diurnal rhythms.

A Zur-mediated transcriptional regulation of the zinc export system in Pseudomonas aeruginosa.

The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of Pseudomonas aeruginosa. The transcription of its many Zn transporters is therefore tightly regulated by a known set of transcription factors involved in either the import or the export of Zn. In this work, we show that the Zur protein, a well-known repressor of Zn import, plays a dual role and functions in both import and export processes. In a situation of Zn excess, Zur represses Zn entry, but also activates the transcription of czcR, a positive regulator of the Zn export system. To achieve this, Zur binds at two sites, located by DNA footprinting in the region downstream the czcR transcription start site. In agreement with this regulation, a delay in induction of the efflux system is observed in the absence of Zur and Zn resistance is reduced. The discovery of this regulation highlights a new role of Zur as global regulator of Zn homeostasis in P. aeruginosa disclosing an important link between Zur and zinc export.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

Dual role of a (p)ppGpp- and (p)ppApp-degrading enzyme in biofilm formation and interbacterial antagonism.

The guanosine nucleotide-based second messengers ppGpp and pppGpp (collectively: (p)ppGpp) enable adaptation of microorganisms to environmental changes and stress conditions. In contrast, the closely related adenosine nucleotides (p)ppApp are involved in type VI secretion system (T6SS)-mediated killing during bacterial competition. Long RelA-SpoT Homolog (RSH) enzymes regulate synthesis and degradation of (p)ppGpp (and potentially also (p)ppApp) through their synthetase and hydrolase domains, respectively. Small alarmone hydrolases (SAH) that consist of only a hydrolase domain are found in a variety of bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa. Here, we present the structure and mechanism of P. aeruginosa SAH showing that the enzyme promiscuously hydrolyses (p)ppGpp and (p)ppApp in a strictly manganese-dependent manner. While being dispensable for P. aeruginosa growth or swimming, swarming, and twitching motilities, its enzymatic activity is required for biofilm formation. Moreover, (p)ppApp-degradation by SAH provides protection against the T6SS (p)ppApp synthetase effector Tas1, suggesting that SAH enzymes can also serve as defense proteins during interbacterial competition.

Role of Two-Component System Networks in Pseudomonas aeruginosa Pathogenesis.

Two-component systems (TCS) are the largest family of signaling systems in the bacterial kingdom. They enable bacteria to cope with a wide range of environmental conditions via the sensing of stimuli and the transduction of the signal into an appropriate cellular adaptation response. Pseudomonas aeruginosa possesses one of the richest arrays of TCSs in bacteria and they have been the subject of intense investigation for more than 20 years. Most of the P. aeruginosa TCSs characterized to date affect its pathogenesis, via the regulation of virulence factors expression, modulation of the synthesis of antibiotic/antimicrobial resistance mechanisms, and/or via linking virulence to energy metabolism. Here, we give an overview of the current knowledge on P. aeruginosa TCSs, citing key examples for each of the above-mentioned regulatory actions. We then conclude by mentioning few small molecule inhibitors of P. aeruginosa TCSs that have shown an antimicrobial action in vitro.

Epidemiology and associated factors of polypharmacy in older patients in primary care: a northern Italian cross-sectional study.

BACKGROUND: A precondition for developing strategies to reduce polypharmacy and its well-known harmful consequences is to study its epidemiology and associated factors. The objective of this study was to analyse the prevalence of polypharmacy (defined as ≥8 prescribed drugs), of potentially inappropriate medications (PIMs) and major drug-drug interactions (DDIs) among community-dwelling general practice patients aged ≥75 years and to identify characteristics being associated with polypharmacy. METHODS: This cross-sectional study is derived from baseline data (patients' demographic/biometric characteristics, diagnoses, medication-related data, cognitive/affective status, quality of life) of a northern-Italian cluster-RCT. PIMs and DDIs were assessed using the 2012 Beers criteria and the Lexi-Interact® database. Data were analysed using descriptive methods, Wilcoxon rank-sum tests, Fisher's exact tests and Spearman correlations. RESULTS: Of the eligible patients aged 75+, 13.4% were on therapy with ≥8 drugs. Forty-three general practitioners and 579 patients participated in the study. Forty five point nine percent of patients were treated with ≥1 Beers-listed drugs. The most frequent PIMs were benzodiazepines/hypnotics (19.7% of patients) and NSAIDs (6.6%). Sixty seven point five percent of patients were exposed to ≥1 major DDI, 35.2% to ≥2 major DDIs. Antithrombotic/anticoagulant medications (30.4%) and antidepressants/antipsychotics (23.1%) were the most frequently interacting drugs. Polypharmacy was significantly associated with a higher number of major DDIs (Spearman's rho 0.33, p < 0.001) and chronic conditions (Spearman's rho 0.20, p < 0.001), higher 5-GDS scores (thus, lower affective status) (Spearman's rho 0.12, p = 0.003) and lower EQ-5D-5L scores (thus, lower quality of life) (Spearman's rho - 0.14, p = 0.001). Patients' age/sex, 6-CIT scores (cognitive status), BMI or PIM use were not correlated with the number of drugs. CONCLUSIONS: The prevalence of polypharmacy, PIMs and major DDIs was considerable. Results indicate that physicians should particularly observe their patients with multiple conditions, reduced health and affective status, independently from other patients' characteristics. Careful attention about indication, benefit and potential risk should be paid especially to patients on therapy with specific drug classes identified as potentially inappropriate or prone to major DDIs in older persons (e.g., benzodiazepines, NSAIDs, protonic pump inhibitors, antithrombotics/anticoagulants, antidepressants/antipsychotics). TRIAL REGISTRATION: The cluster-RCT on which this cross-sectional analysis is based was registered with Current Controlled Trials Ltd. (ID ISRCTN: 38449870 ) on 2013-09-11.

Expert-based medication reviews to reduce polypharmacy in older patients in primary care: a northern-Italian cluster-randomised controlled trial.

BACKGROUND: Evidence regarding clinically relevant effects of interventions aiming at reducing polypharmacy is weak, especially for the primary care setting. This study was initiated with the objective to achieve clinical benefits for older patients (aged 75+) by means of evidence-based reduction of polypharmacy (defined as ≥8 prescribed drugs) and inappropriate prescribing in general practice. METHODS: The cluster-randomised controlled trial involved general practitioners and patients in a northern-Italian region. The intervention consisted of a review of patient's medication regimens by three experts who gave specific recommendations for drug discontinuation. Main outcome measures were non-elective hospital admissions or death within 24 months (composite primary endpoint). Secondary outcomes were drug numbers, hospital admissions, mortality, falls, fractures, quality of life, affective status, cognitive function. RESULTS: Twenty-two GPs/307 patients participated in the intervention group, 21 GPs/272 patients in the control group. One hundred twenty-five patients (40.7%) experienced the primary outcome in the intervention group, 87 patients (32.0%) in the control group. The adjusted rates of occurrence of the primary outcome did not differ significantly between the study groups (intention-to-treat analysis: adjusted odds ratio 1.46, 95%CI 0.99-2.18, p = 0.06; per-protocol analysis: adjusted OR 1.33, 95%CI 0.87-2.04, p = 0.2). Hospitalisations as single endpoint occurred more frequently in the intervention group according to the unadjusted analysis (OR 1.61, 95%CI 1.03-2.51, p = 0.04) but not in the adjusted analysis (OR 1.39, 95%CI 0.95-2.03, p = 0.09). Falls occurred less frequently in the intervention group (adjusted OR 0.55, 95%CI 0.31-0.98; p = 0.04). No significant differences were found regarding the other outcomes. Definitive discontinuation was obtained for 67 (16.0%) of 419 drugs rated as inappropriate. About 6% of the prescribed drugs were PIMs. CONCLUSIONS: No conclusive effects were found regarding mortality and non-elective hospitalisations as composite respectively single endpoints. Falls were significantly reduced in the intervention group, although definitive discontinuation was achieved for only one out of six inappropriate drugs. These results indicate that (1) even a modest reduction of inappropriate medications may entail positive clinical effects, and that (2) focusing on evidence-based new drug prescriptions and prevention of polypharmacy may be more effective than deprescribing. TRIAL REGISTRATION: Current Controlled Trials (ID ISRCTN: 38449870), date: 11/09/2013.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences.

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP) has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles.

Correction: The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

[This corrects the article DOI: 10.1371/journal.pgen.1006354.].

Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria.

The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission.

Caught at its own game: regulatory small RNA inactivated by an inducible transcript mimicking its target.

A relevant, yet little recognized feature of antisense regulation is the possibility of switching roles between regulatory and regulated RNAs. Here we show that induction of a Salmonella gene relies on the conversion of a small RNA from effector to regulatory target. The chiP gene (formerly ybfM), identified and characterized in the present study, encodes a conserved enterobacterial chitoporin required for uptake of chitin-derived oligosaccharides. In the absence of inducer, chiP is kept silent by the action of a constitutively made small RNA, ChiX (formerly SroB, RybC), which pairs with a sequence at the 5' end of chiP mRNA. Silencing is relieved in the presence of chitooligosaccharides due to the accumulation of an RNA that pairs with ChiX and promotes its degradation. Anti-ChiX RNA originates from an intercistronic region of the chb operon, which comprises genes for chitooligosaccharide metabolism and whose transcription is activated in the presence of these sugars. We present evidence suggesting that the chb RNA destabilizes ChiX sRNA by invading the stem of its transcription terminator hairpin. Overall, our findings blur the distinction between effector and target in sRNA regulation, raising the possibility that some of the currently defined targets could actually be inhibitors of sRNA function.

The diguanylate cyclase SadC is a central player in Gac/Rsm-mediated biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen and a threat for immunocompromised and cystic fibrosis patients. It is responsible for acute and chronic infections and can switch between these lifestyles upon taking an informed decision involving complex regulatory networks. The RetS/LadS/Gac/Rsm network and the cyclic-di-GMP (c-di-GMP) signaling pathways are both central to this phenomenon redirecting the P. aeruginosa population toward a biofilm mode of growth, which is associated with chronic infections. While these two pathways were traditionally studied independently from each other, we recently showed that cellular levels of c-di-GMP are increased in the hyperbiofilm retS mutant. Here, we have formally established the link between the two networks by showing that the SadC diguanylate cyclase is central to the Gac/Rsm-associated phenotypes, notably, biofilm formation. Importantly, SadC is involved in the signaling that converges onto the RsmA translational repressor either via RetS/LadS or via HptB/HsbR. Although the level of expression of the sadC gene does not seem to be impacted by the regulatory cascade, the production of the SadC protein is tightly repressed by RsmA. This adds to the growing complexity of the signaling network associated with c-di-GMP in P. aeruginosa. While this organism possesses more than 40 c-di-GMP-related enzymes, it remains unclear how signaling specificity is maintained within the c-di-GMP network. The finding that SadC but no other diguanylate cyclase is related to the formation of biofilm governed by the Gac/Rsm pathway further contributes to understanding of this insulation mechanism.

Hierarchical management of carbon sources is regulated similarly by the CbrA/B systems in Pseudomonas aeruginosa and Pseudomonas putida.

The CbrA/B system in pseudomonads is involved in the utilization of carbon sources and carbon catabolite repression (CCR) through the activation of the small RNAs crcZ in Pseudomonas aeruginosa, and crcZ and crcY in Pseudomonas putida. Interestingly, previous works reported that the CbrA/B system activity in P. aeruginosa PAO1 and P. putida KT2442 responded differently to the presence of different carbon sources, thus raising the question of the exact nature of the signal(s) detected by CbrA. Here, we demonstrated that the CbrA/B/CrcZ(Y) signal transduction pathway is similarly activated in the two Pseudomonas species. We show that the CbrA sensor kinase is fully interchangeable between the two species and, moreover, responds similarly to the presence of different carbon sources. In addition, a metabolomics analysis supported the hypothesis that CCR responds to the internal energy status of the cell, as the internal carbon/nitrogen ratio seems to determine CCR and non-CCR conditions. The strong difference found in the 2-oxoglutarate/glutamine ratio between CCR and non-CCR conditions points to the close relationship between carbon and nitrogen availability, or the relationship between the CbrA/B and NtrB/C systems, suggesting that both regulatory systems sense the same sort or interrelated signal.

Catabolite repression in Pseudomonas aeruginosa†PAO1 regulates the uptake of C4 -dicarboxylates depending on succinate concentration.

In Pseudomonas aeruginosa carbon catabolite repression (CCR) is exerted by the CbrA/B-CrcZ-Crc global regulatory system. Crc is a translational repressor that, in the presence of preferred carbon sources, such as C4 -dicarboxylates, impairs the utilization of less preferred substrates. When non-preferred substrates are present, the CrcZ sRNA levels increase leading to Crc capture, thereby allowing growth of the bacterium at the expense of the non-preferred substrates. The C4 -dicarboxylate transport (Dct) system in P. aeruginosa is composed of two main transporters: DctA, more efficient at mM succinate concentrations, and DctPQM, more important at µM. In this study, we demonstrate that the Dct transporters are differentially regulated by Crc, depending on the concentration of succinate. At high concentrations, Crc positively regulates the expression of the dctA transporter gene and negatively regulates dctPQM post-transcriptionally. The activation of dctA is explained by a Crc-mediated repression of dctR, encoding a transcriptional repressor of dctA. At low succinate concentrations, Crc regulation is impaired. In this condition, CrcZ levels are higher and therefore more Crc proteins are sequestered, decreasing the amount of Crc available to perform CCR on dctR and dctPQM. As a result, expression of dctA is reduced and that of dctPQM is increased.

Identification and characterisation of G-quadruplex DNA-forming sequences in the Pseudomonas aeruginosa genome.

A number of Gram-negative bacteria such as Pseudomonas aeruginosa are becoming resistant to front-line antibiotics. Consequently, there is a pressing need to find alternative bio-molecular targets for the development of new drugs. Since non-canonical DNA structures such as guanine-quadruplexes (G4s) have been implicated in regulating transcription, we were interested in determining whether there are putative quadruplex-forming sequences (PQS) in the genome of Pseudomonas aeruginosa. Using bioinformatic tools, we screened 36 genes potentially relevant to drug resistance for the presence of PQS and 10 of these were selected for biophysical characterisation (i.e. circular dichroism and thermal difference UV/Vis spectroscopy). These studies showed that three of these G-rich sequences (linked to murE, ftsB and mexC genes) form stable guanine-quadruplexes which were studied by NMR spectroscopy; detailed analysis of one of the sequences (mexC) confirmed that it adopts a two-quartet antiparallel quadruplex structure in the presence of K+ ions. We also show by FRET melting assays that small molecules can stabilise these three new G4 DNA structures under physiological conditions. These initial results could be of future interest in the development of new antibiotics with alternative bio-molecular targets which in turn would help tackle antimicrobial resistance.

Mechanism of inhibition of bacterial RNA helicases by diazo dyes and implications for antimicrobial drug development.

RNA helicases represent attractive drug targets as their activity is linked to several human diseases and impacts microbial infectious processes. While some inhibitors of human RNA helicases demonstrated therapeutic potential as anticancer and antiviral drugs in preclinical trials, chemical inhibition of microbial RNA helicases is less investigated. Here, we address this matter by focusing on the RhlE proteobacterial group of RNA helicases. Having previously shown that the RhlE2 RNA helicase is important for the virulence of the opportunistic pathogen Pseudomonas aeruginosa, we screened a library of 1280 molecules for inhibitors of RhlE2 RNA-dependent ATP hydrolytic activity. The most potent inhibitor is the diazo compound Chicago Sky Blue (CSB). Using hydrogen-deuterium exchange mass spectrometry and biochemical assays, we mapped CSB binding to RhlE2 catalytic core and defined its inhibitory mechanism. Targeting microbial RNA helicases as therapeutic strategy is challenging due to potential side-effects linked to protein conservation across life kingdoms. Interestingly, our structure-activity relationship analysis delineates other diazo dyes closely related to CSB differentially affecting RhlE homologs. Our work could thus be exploited for future drug development studies, which are extremely timely considering the increasing spread of antibiotic resistance among bacterial pathogens.

Identification of C(4)-dicarboxylate transport systems in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa utilizes preferentially C(4)-dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C(4)-dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA(PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C(4)-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C(4)-dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C(4)-dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.