Introduction

Bacteria sense environmental changes by perceiving extracellular hints such as the concentration of nutrients for example, carbon, nitrogen, phosphate, iron, sulphur, and growth conditions for instance pH, temperature, oxygen availability, osmotic stress and survive in response to these perturbations (Shimizu 2014). The environmental perturbations send signal to the cells sensing the change which induces the secretion of auto-inducer molecules leading to the sensitisation of whole community. This allows the bacterial community to synchronise gene expression, and thereby carry out collective activities for growth and survival. Induction of changes sensed by individual cells may lead to changes in the whole community, through quorum sensing. The signals provided to the transcriptional regulatory systems shape the physiological and morphological adjustments that facilitate effective adaptation and survival of the whole bacterial community (Seshasayee 2006). This metabolic versatility shapes the ecological fitness of an organism by coordinating a number of distinctive global regulatory networks leading to expression (Shimizu 2014).

In a face-changing environment where numerous carbon sources are available at concentrations that define the survival of microorganisms, bacterial species activate universal regulation systems that synchronise metabolism. The presence of variety of substrates in a natural environment allows bacteria to either co-utilise carbon sources or utilise preferential ones that are most effective for growth. The selection of a preferred carbon source over others and inhibition of the uptake machinery or genes essential for the catabolism of non-preferred ones at the same time is regulated precisely. The regulated selection of preferred carbon source over non-preferred ones is termed as carbon catabolite repression (CCR). Originally CCR was illustrated in Escherichia coli for utilisation of sugars in a hierarchical manner (Magasanik 1970). CCR can be carried out in two ways: first, inhibition of expression of enzymes of the pathways for non-preferred carbon source and second, reshuffling of metabolism for activation of appropriate genes leading to the assimilation of preferred carbon sources (Rojo 2010). Being a complex mechanism, CCR can be arbitrated by various regulatory systems. The fundamental molecular regulatory networks differ among individual bacterial groups. The underlying machinery and the regulatory partners required for CCR have been studied extensively in Enterobacteria, Firmicutes and in Pseudomonas (reviewed in Gorke and Stulke 2008).

CCR in Pseudomonas: the general mechanism

Pseudomonas is a genus of ubiquitous bacteria found in a wide range of habitats such as soil, water, animals and plant influencing them either positively or negatively. Some well-known examples include opportunistic animal pathogen P. aeruginosa (Sonnleitner et al. 2017); plant pathogen P. syringiae (Chakravarthy et al. 2017; Filiatrault et al. 2013); bioremediation agent P. putida (Basu et al. 2006) and P. fluorescens as plant growth promoting and bio-control agent (Liu et al. 2017). Pseudomonas is well adapted to various environments as they can utilise a wide array of carbon sources (Dos Santos et al. 2004; Molina et al. 2000) but are reported to utilise a limited number of sugars such as glucose, glucuronic acid and fructose (Daddaoua et al. 2009; Silby et al. 2011). The metabolic patterns in these microbes also govern a lifestyle that acclimatises to environmental conditions where sugars are in limited concentrations. Glucose utilisation machinery is organised in three-tiered metabolic system that generates 6-phosphogluconate and has remarkable differences as compared to Escherichia coli and Bacillus species. The glucose is transported and metabolised through the phosphoenolpyruvate phosphotransferase system (PTS) in these bacteria whereas in Pseudomonas, glucose crosses the outer membrane into the periplasmic space through the OprB-1 porin and is directly transported into the cell or can be oxidised to gluconate or 2-ketogluconate in the periplasmic space. All the metabolites produced during periplasmic glucose oxidation have their own transporters through which they can be transported inside the cell. Once imported inside the cell, glucose, gluconate and 2-ketogluconate are oxidised through the Entner–Doudoroff pathway (Del Castillo et al. 2007). Differences in transport of glucose also influence the regulation of catabolism in Pseudomonas. In E. coli, the PTS, a multi-protein phosphorylation cascade helps in glucose uptake and metabolism. Whereas in Pseudomonas, glucose metabolism is regulated by one-component systems (OCSs) HexR, PtxS, PtxR and GntR transcriptional factors as well as two-component system (TCS) GltR/GtrS transcriptional factor which are induced by intermediates of the ED pathway (Udaondo et al. 2018).

Escherichia coli utilises glucose as a preferred carbon source suppressing catabolic pathways of glycerol, organic acids and amino acids by CCR (Postma et al. 1993). On the other hand, metabolism of glucose is under catabolic repression in Pseudomonas because organic acids are preferred over carbohydrates. The repression in Pseudomonas is reverse of the phenomenon followed in enteric bacteria and it is often referred to as “reverse CCR”. For instance, presence of succinate and glucose creates CCR conditions where expression of glucose catabolism enzymes of P. aeruginosa was inhibited until succinate was available (Collier et al. 1996). This phenomenon of physiological hierarchy to nutrient accessibility that leads to the preferential utilisation of succinate over other carbon sources for growth is termed as succinate mediated catabolite repression (SMCR). Organic acids succinate and acetate are known to repress enzymes for utilisation of gluconate, glycerol, fructose and mannitol. The sequential hierarchy among amino acid utilisation has also been reported in P. putida (Hester et al. 2000a, b; Moreno 2007; Rojo 2010). P. putida strain CSV86 has unique ability to utilise aromatic compounds such as naphthalene prior to glucose where succinate does not limit naphthalene degradation but impairs transport system and enzymes for glucose assimilation (Basu et al. 2006, 2007). In presence of succinate and benzoate, benzoate was preferentially metabolised while glucose transport and its metabolism were suppressed in P. putida CSV86 (Choudhary et al. 2017).

The CCR mediators

At present, the regulatory system of CCR in Pseudomonas encompasses the following components: Crc and Hfq proteins, the TCS: CbrAB and sRNAs: CrcY/CrcZ. The subsequent sections of the review summarise how these components cooperate and function to execute CCR.

Crc protein: the global regulator

Catabolite repression control (Crc) protein was originally reported in P. aeruginosa during random mutagenesis experiments intended to find catabolite repression relieved mutants of amidase (MacGregor 1991; Wolff 1991). Some crc mutants were relieved of repression imparted by succinate over mannitol and glucose transport and enzymes involved in their catabolism. The Crc protein shares up to 25 to 32% sequence homology with DNA repair enzymes of other bacteria but does not show any endonuclease or DNA binding ability (MacGregor 1996). In P. putida and P. aeruginosa, Crc regulates induction of bkd operons for branched-chain keto acid dehydrogenases. Consequently, in several other Pseudomonas species, Crc was described as the regulator of catabolite repression of branched-chain keto acid dehydrogenase (Hester et al. 2000a) enzyme for degradation of alkane (Yuste and Rojo 2001), toluene (Aranda-Olmedo 2005), aromatic compounds (Morales et al. 2004) and biofilm formation (O'Toole et al. 2000). The difference in growth conditions was shown to have differential impact on the expression of crc (Ruiz-Manzano et al. 2005). In P. putida, Crc binds to benR and alkS mRNAs and inhibit the translation leading to repression of benzoate and alkane degradation genes respectively (Moreno 2007, 2008). However, the crystal structure of Crc from P. aeruginosa lacked nuclease activity (Milojevic et al. 2013). This suggested that there must be some other proteins helping the regulatory action being carried out during CCR. Crc exerts a positive impact on the type III secretion system (T3SS) in P. aeruginosa where the master regulator ExsA and the Cbr/Crc signalling system regulate T3SS (Dong et al. 2013). Crc is also an up-regulator of rhamnolipid production in P. aeruginosa (Yang et al. 2015). Some recent studies have identified Crc as a regulator of muconate production in P. putida KT2440 where levels of 4-hydroxybenzoate and vanillate were reduced upon crc deletion (Johnson et al. 2017). Virulence of P. syringae pv. Tomato DC3000 was influenced by Crc (Chakravarthy et al. 2017) which was also reported to coordinate the overall response to oxidative stress by means of reorganisation of central metabolism in P. aeruginosa PAO1 (Corona et al. 2018, 2019).

Hfq: the “RNA matchmaker”

The Hfq protein was initially discovered as a host factor essential for replication of bacteriophage Qβ in E. coli (August et al. 1970). Hfq is a hexameric protein belonging to the class of Sm-family of RNA binding proteins in many bacteria (Zhang et al. 2002) and has a proximal and distal RNA binding site (Sauer and Weichenrieder 2011). Hfq is a regulator in Gram-negative bacteria that assists the regulatory RNA mediated post-transcriptional gene regulation during CCR (Sonnleitner and Blasi 2014; Kambara et al. 2018). During CCR in P. aeruginosa, the translation of target transcripts was inhibited directly by Hfq-Crc complex. Crc bound Hfq on its distal part binds to the A-rich motifs on target mRNA near to ribosome binding site (RBS) and regulates the translation (Sonnleitner et al. 2018b).

Being a global regulator, Hfq interacts with sRNAs ranging in size from 37 to 500 nucleotide and stimulate or restrain mRNA translation through positive or negative regulatory mechanisms (Santiago‐Frangos and Woodson 2018). The size, conformation and combination of complementary sequences of RNA differ when Hfq mediated regulation is considered. During Hfq-dependent regulation, mRNA and sRNA bind to Hfq on different sites. It has been reported that Hfq binding to the mRNA with or without sRNA influence the fate of mRNAs. For example, during negative regulation, Hfq assists stabilisation and recruitment of sRNA and help to recruit ribonucleases which ultimately degrade mRNAs. In positive regulation sRNAs and Hfq binding stabilises mRNA by changing mRNA folding which allows ribosome access or blocking access of a ribonuclease to protect the mRNA (Kavita et al. 2018). The RNA ligands are highly varied in nature which is reflected in cognate protein partners that comprise of an array of functions related to various enzymes (Butland et al. 2005). Hfq functions as a stabiliser of regulatory sRNAs like CrcZ and CrcX in the absence of their target transcripts by facilitating base-pairing to the mRNAs (Zhang et al. 2002); it helps in the repression of translation and also mRNA activation (Gottesman and Storz 2011; Vogel and Luisi 2011). Thus, the selectivity of substrate for Hfq has to be flexible enough to cooperate with a range of RNA-protein complexes to find a perfect match (Updegrove et al. 2016; Kavita et al. 2018; Santiago-Frangos and Woodson 2018).

The function of Hfq has been established by generation of Hfq deletion strain of P. aeruginosa which influences antibiotic susceptibility, energy metabolism, cell wall composition and the levels of c-di-GMP (Sonnleitner et al. 2018b). In P. aeruginosa PAO1, Hfq represses three mRNAs namely amiE (encoding an aliphatic amidase), estA (esterase) and phzM (phenazine-specific methyltransferase) (Sonnleitner and Blasi 2014). The translational inhibition applied by Crc operates in association with the Hfq protein to reduce translation of DmpR regulator of the Dmp-pathway (dimethylphenol) for catabolism of phenol in P. putida (Wirebrand et al. 2018). Functional characterisation of Acinetobacter baumannii lacking Hfq revealed its role in environmental adjustment and virulence by amending stress responses, morphology and virulence factors (Kuo et al. 2017). In Azotobacter vinelandii, the gene gluP encoding glucose transporter has A-rich Hfq-binding motif and thus glucose transport is under CCR through Crc/Hfq and CbrA/CbrB regulatory systems (Quiroz-Rocha et al. 2017). Hfq protein has a pivotal role as a pleiotropic regulator mediating complex CCR regulation that influences metabolism, virulence, quorum sensing and stress response in Pseudomonas and related bacterial species.

CrcZ, CrcY: the regulatory sRNAs

The role of small RNAs (sRNAs) (~ 50–300 nucleotides) has been determinedly established as important regulators of metabolism in bacteria. The sRNAs are known for regulating bacterial environmental response pathways and the most comprehensively studied sRNAs are known to regulate mRNAs of target genes by defective and short base pairing as trans-encoded sRNAs. Some sRNAs bind at or near the RBS and block translation by obstructing ribosomes. Additional types of sRNAs bind far-away from the target site and interfere with ribosome binding through either inhibiting formation of secondary structures or altering mRNA stability (Storz et al. 2011). The group of sRNAs that sequester and titrate the RNA binding proteins of CsrA/RsmA family control translation initiation. These small proteins explicitly bind to GGA trinucleotide situated in the 5′ leader sequence of target mRNAs and inhibit their translation.

One such protein-RNA system is the CrcZ/Crc system that works in catabolite repression control in Pseudomonas species. In conditions where CCR is operated, Crc protein binds to AC rich motifs within or adjacent to RBS of target mRNAs inhibiting their translation. When CCR is relieved, the regulatory RNA CrcZ, CrcY/CrcZ and CrcZ/CrcX in P. aeruginosa PAO1, P. putida, and P. syringae respectively are projected to bind and trap the protein (Sonnleitner et al. 2009; Moreno et al. 2012; Filiatrault et al. 2013). However, the structural and biochemical studies posed a question against the role of Crc mediated CCR in P. aeruginosa PAO1 as purified Crc was neither able to bind the aliphatic amidase encoding amiE mRNA (target mRNA) nor to CrcZ sRNA (Milojevic et al. 2013). Numerous genes of P. aeruginosa involved in catabolic pathways are known to be inhibited post-transcriptionally by Hfq by binding on the distal face of Hfq during growth on succinate (Sonnleitner et al. 2018b). The Hfq binds to CrcZ and Crc protein which as a complex exert regulation (Rojo 2010; Sonnleitner and Blasi 2014). The response of a bacterium to a particular signal is influenced by the concurrent cellular environment of Hfq-dependent sRNAs, Crc protein, target mRNAs and the capability of these RNAs to compete with each other so that precise regulatory pathways are arranged on a priority basis (Santiago‐Frangos and Woodson 2018). To understand the interdependence of Hfq-sRNA mediated control in P. aeruginosa, the possibility whether the regulatory RNA CrcZ can interfere with riboregulation mediated by the sRNAs PrrF1-2 was checked. antR mRNA encoding a transcriptional activator of the antABC operon is required for anthranilate degradation and is known to be controlled by PrrF1-2 sRNA. CrcZ cross-regulates mRNAs of antR with Hfq-mediated riboregulation. In iron limitation and growth on preferred carbon source, antR translation was repressed by PrrF1-2 and Hfq. In non-CCR conditions, CrcZ competes for Hfq binding which interferes with PrrF1-2 binding, activating antR translation and degradation of anthranilate (Sonnleitner et al. 2017).

CbrA-CbrB: the two-component system

The immense versatility and ability to quickly adapt to the fluctuating nutritional environment is due to the presence of well-regulated two-component systems (TCSs) programmed in genomes of bacteria. The TCSs encompasses a sensor kinase (SK) and a response regulator (RR). The autophosphorylation caused in the histidine kinase (HK) domain of the SK is induced by environmental stimulus leading to subsequent transfer of phosphate to the acceptor domain of RR triggering activation of regulator protein (von Bodman et al. 2008). Thus, activated RR stimulates expression of genes critical for adjustment towards the altered environmental condition. This adaptation of bacterial cells through activation of TCS leads to change in physiological functions aiding utilisation of alternative sources when certain nutrients become limiting. When nitrogen or phosphate is limiting the NtrB-NtrC and the PhoR-PhoB are activated for nitrogen and phosphate assimilation, respectively (Bourret and Silversmith 2010). Generally, the N-terminal periplasmic domain of SK senses nutrients or external stimuli priming the activation of respective genes by the RR. Another TCS known as CbrAB has ability to regulate carbon and nitrogen catabolism in Pseudomonas and plays a global regulatory role (Nishijyo et al. 2001; Zhang and Rainey 2008). In CbrAB TCS, CbrA is HK and CbrB is RR that regulates and establishes a healthy carbon/nitrogen balance (Sonnleitner et al. 2009, 2012b; Moreno et al. 2012). CbrB is an NtrC family RR that activates the transcription of sRNAs (Nishijyo et al. 2001; Sonnleitner et al. 2012a). The CbrA/CbrB system has several functions in Pseudomonas species such as regulation of swarming motility, biofilm formation, antibiotic and stress resistance (Amador et al. 2010; Yeung et al. 2011). The phenotypic analysis in CbrAB mutants of P. fluorescens SBW25 resulted in growth deficiency on various carbohydrates and amino acids (Zhang and Rainey 2008). It was also found that the leucine metabolism in P. aeruginosa is also under the influence of CbrB/Crc regulation. Leucine is metabolised by enzymes encoded in the liuRABCDE gene cluster, where LiuR is the regulator. In the presence of leucine, LiuD was strongly expressed but repressed in the presence of glucose or succinate. These results indicate that in absence of Crc, LiuD expression was independent of the presence of carbon source, however, its expression was impaired in cbrB- mutant (Díaz-Pérez et al. 2018). The mutants of cbrAB of opportunistic human pathogen P. aeruginosa PAO1 also had growth defects (Nishijyo et al. 2001). CbrAB along with Crc regulates the carbohydrate metabolism as well as the amino acid metabolism.

The definite mechanism of CCR in Pseudomonas

In Pseudomonas species, the hierarchy of carbon source utilisation emerges through translational repression mediated by Crc protein. The A-rich sequences near the RBS of the mRNAs are crucial for the repression. Crc does not bind to the target mRNA directly; instead, Hfq allows the binding of the A-rich motifs to its distal face. In presence of a non-preferred carbon source (for example, glucose: when catabolite repression is not necessary), Crc/Hfq repression is relieved by expression of a non-coding RNA-CrcZ or CrcY, bearing various copies of the A-rich motif, which sequester Hfq-Crc complex (Fig. 1a). The presence of non-preferred carbon source in the medium shows a strident increase in the levels of CrcZ/Y leading to sequestration of Crc which allows ribosome binding and translation of the mRNAs for utilisation of non-preferred carbon source (Sonnleitner et al. 2018b).

Fig. 1
figure 1

Mechanism of succinate mediated catabolite repression (SMCR) in Pseudomonas. a In the non-CCR conditions (e.g. in presence glucose), Crc binds to Hfq bound sRNA (CrcZ/CrcY) allowing translation of glucose utilisation genes. b Under the CCR conditions (SMCR), when preferred and non-preferred carbon source (succinate and glucose respectively) are both present, the Crc-Hfq complex binds to the RBS inhibiting translation of glucose utilisation genes. The repression is relived (under non-CCR conditions) via sequestering of Crc and Hfq by CrcZ whose expression is activated by CbrAB two-component system and CrcY whose expression is controlled by unknown signals. This model is based on the results of the earlier studies (Kavita et al. 2018; Rojo 2010; Sonnleitner and Blasi 2014; Sonnleitner et al. 2018a, b). A green or red arrow indicates activation or inhibition respectively

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In presence of a favored carbon source (for example, succinate), the concentration of CrcZ/Y depletes and free Crc possibly remains at a concentration high enough to form complex with Hfq that binds to the target mRNAs holding back their translation (Fig. 1b) and thus inhibiting utilisation of non-preferred carbon sources. The level of CrcZ is regulated by CbrAB which is activated to set the hierarchy of carbon source utilisation. Inactivation of CbrB does not show any influence on CrcY expression as CrcY activation relies largely on systems different from CbrAB. Figure 1 illustrates the mechanism of CCR in Pseudomonas species and involvement of its mediators- Crc and Hfq proteins, CrcZ/Y and CbrAB. The physiological traits that are governed by Crc, Hfq CrcZ/Y and CbrAB in various bacterial species are listed in Table 1.

Table 1 Crc, Hfq and sRNA mediated regulation in various bacterial species
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Influence of CCR on catabolic pathways of Pseudomonas

The activation of a catabolic pathway depends on the combined activity of several global regulators in response to external or internal signals generated due to the cell’s need to adapt and survive. The CCR interferes with the stimulation of certain catabolic pathways by employing mediators for down-regulation of particular transcriptional regulators or by interfering with the ability of the regulators to control transcription. Rojo (2010) has extensively reviewed influence of CCR on the degradation of alkane, toluene/xylene and phenol degradation pathways encoded by OCT, Tol and pVI150 plasmids respectively in P. putida.

Influence of CCR on virulence

In P. aeruginosa which is a well known human pathogen, expression of virulence genes is governed by CCR. A pathogenic bacterium infects the host, with the prime aim of acquiring nutrients to survive in the environment. Therefore it looks for alternative carbon sources and therefore the metabolism is influenced by CCR. Various virulence systems are known to amend the pathogenicity of P. aeruginosa. One of the important mechanisms for virulence regulation is quorum sensing (QS) which employs secretion of small diffusible signal molecules in surrounding environment in order to synchronise gene expression and concerted behaviour, for example biofilm formation (Miller and Bassler 2001). It has been reported that the Pseudomonas quinolone signalling (pqs) is affected by Crc in a nutrient-dependent manner (Zhang et al. 2014). The RhlAB system is known for rhamnolipid synthesis which is a virulence factor required during P. aeruginosa lung infection. Several proteases and chaperones together serve to maintain quality control of cellular proteins that are known as the protein quality control (PQC) systems. The PQC of Gram-negative bacteria has Lon and ClpX (clp, caseinolytic protease) which are ATP-dependent proteases (Mogk and Bukau 2006). Crc induces Rhl production by down-modulation of Lon protease. The hfq and crc deletion leads to reduced production of the QS signal molecule N-Butyryl-homoserine lactone (C4-HSL) in P. aeruginosa (Sonnleitner et al. 2006; Yang et al. 2015). The rhl QS signal C4-HSL production was balanced by disruption of Lon protease which is influenced by crc deletion. It was also reported that the repression of rhl QS by ClpX was Lon independent. In addition, ClpP protease is a negative regulator of rhl QS therefore, ClpPX in combination have a role in linking CCR and PQC mediated regulation in P. aeruginosa (Yang and Lan 2016).

Influence of CCR on antibiotic resistance

Apart from virulence, CCR also plays an important role in the development of antibiotic resistance. A steady rise in drug resistance among the clinical isolates poses a challenge towards treatment of various infectious diseases. It becomes important to understand the molecular mechanism of drug resistance in depth to enhance sensitivity of bacteria to the antibiotics in use. In P. aeruginosa antibiotics, amino-acids and carbon sources share same ports of entry for getting inside the cell and the transporter encoding genes are frequently controlled by CCR. In P. aeruginosa defects in type III secretion and motility due to Crc deletion were reported. The mutant strain became more susceptible to antibiotics such as beta-lactams, aminoglycosides, fosfomycin and rifampicin (Linares et al. 2010). The hfq deletion in P. aeruginosa strains also lead to increased susceptibility towards various antibiotics. During CCR i.e. by adding non-preferred carbon source, the expression of CrcZ increased sequestering Hfq which lead to enhanced antibiotic sensitivity (Sonnleitner et al. 2018a). Thus, CCR mediators can be great targets for generating ways for making P. aeruginosa more sensitive to different classes of antibiotics.

Influence of CCR on bioremediation pathways

The molecular means of CCR are important in understanding the behaviour of bacterial species in the environment where they degrade xenobiotic compounds. These compounds are the ones whose concentrations are built up as they are difficult to degrade and create environmental pollution. A number of strains of Pseudomonas had been reported for bioremediation potential in laboratory condition (reviewed in Das and Chandran 2011). However, degradation of any of these compounds may be influenced by the presence of multiple carbon sources in natural environment. Contamination by aromatic compounds poses environmental problems with delayed degradation as they are not the preferred carbon compounds for majority of bacteria. It has been reported that succinate being a preferred carbon source, represses degradation of benzoate and alkanes in laboratory conditions, where the global regulator Crc has a role to play (Wang and Shao 2013). Crc is known to be involved in CCR caused by succinate that leads to repression of a number of genes involved in sugar metabolism in both P. aeruginosa (Collier et al. 1996; MacGregor 1996) and P. putida (Hester et al. 2000a, b). Crc modulates alkane degradation pathway encoded in the OCT plasmid of P. putida with varying levels of Crc in accordance with the growth conditions (Ruiz-Manzano et al. 2005). When P. putida was grown in association with plant roots, the root exudates reduced phenanthrene degradation (Rentz et al. 2004). This is suggestive of an important fact that CCR may be well operational in natural environments where concentrations of carbon sources are often limiting leading to hierarchical utilisation or repression of certain carbon sources. An interesting approach was suggested wherein the influence of trace amounts of preferred substrate was checked. Use of low concentrations of succinate in P. putida mt-2 batch culture improved the toluene removal efficiency (Tsipa et al. 2017). Similarly, strategies for bioremediation of other xenobiotics compounds can be optimised using an in-depth understanding of the molecular networks of CCR and mediators to generate a superior bioremediation agent.

Influence of CCR on plant growth promotion pathways

Microbial strains present in the rhizosphere that can improve growth of plants are known as plant growth promoting rhizobacteria (PGPR) (Kloepper et al. 1980). The PGPR have been explored as bio-inoculants in the fields for several decades. It would be highly desirable to develop multi-trait PGPR strains that can fix nitrogen, solubilise phosphate, control disease and have other plant beneficial traits. Several PGPR strains are routinely employed but their positive responses to plants are often limited to laboratory or greenhouse and the results are inconsistent in field conditions. The plant roots secrete organic compounds in root exudates that help microbes for survival in the rhizosphere and they in return promote plant growth. The compositions of root exudates vary according to the plant species and often comprise complex mixtures. Presence of various organic acids and sugars in rhizosphere execute CCR for preferential utilisation of carbon sources. This facilitates the bacterium to compete and survive among existing microbial communities. While it is not yet clear how bacteria choose the substrate in a rhizosphere where CCR could be regulating metabolism, various experiments have been conducted in vitro with commonly found organic acids present in the root exudates. For instance, succinate and malate were reported to repress the gluconate production which was shown to be the main mechanism of mineral phosphate solubilisation (MPS) in plant growth promoting fluorescent Pseudomonas (Patel et al. 2011). Similarly, in our laboratory, the biochemical basis of SMCR of MPS in two strains of Klebsiella (SM6 and SM11) was established. The SMCR was operative on glyoxylate shunt enzymes (isocitrate lyase and glyoxylate oxidase) hence leading to repression of oxalate mediated MPS (Rajput et al. 2013). Furthermore, it was found that iclR repressor of aceBAK operon was the key regulator of the MPS repression. Therefore, generation of iclR null mutants in both the strains relieved repression of MPS up to 54% and 59% and improved PGP activities as compared to that of wild type strains even in presence of succinate (Rajput et al. 2015). A similar type of biochemical and molecular basis of SMCR is yet to be established in PGP Pseudomonas species. A strategy of similar kind may be employed when phosphate solubilisation or other PGP traits are influenced by the components of root exudates and CCR. By these means repression relieved strains can be developed which may further improve plant growth under natural soil conditions also. A more comprehensive genome mining effort need to be carried out to unravel the genetic basis underlying the CCR in Pseudomonas and other rhizobacterial species which would be critical for generation of efficient biofertilisation strategies.

Promising genome-wide methodologies for rewiring the functional complexity between Crc, Hfq and sRNAs

The regulated expression of common responses in bacteria is a consequence of simultaneous integration of multiple signals conferring plasticity, versatility and efficiency. To explore the contribution of each and every input to the expression machinery, it is necessary to analyse the post-transcriptional regulation in Pseudomonas species. The Crc protein, Hfq and sRNAs are the main players of the post-transcriptional regulation. To understand the interlaced regulation, a combination of many omics approaches may produce a broad representation of bacterial adaptation to the external milieu.

The potential roles of Crc, Hfq and sRNAs in different bacteria have been characterised by phenomic, transcriptomic and proteomic analyses of knockouts of respective genes. Role of Hfq was established in P. aeruginosa (Sonnleitner et al. 2006), Sinorhizobium meliloti (Torres-Quesada et al. 2010), Brucella melitensis (Cui et al. 2013), Serratia sp. ATCC 39006 (Wilf et al. 2013), E. coli (Bilusic et al. 2014), Clostridium difficile (Boudry et al. 2014), Yersinia pestis (Deng et al. 2014), Bacillus subtilis (Hämmerle et al. 2014) by using omics approaches.

The transcriptome data revealed the influence of Hfq on genes involved in bacterial metabolism suggesting role of Hfq in controlling the metabolic versatility of P. fluorescens. The ribosome profiling experiments were performed to dissect the role of Hfq in regulation at the levels of transcript abundance and translation. The data revealed the negative role of Hfq by controlling translation of mRNAs encoding transport system and enzyme-based systems for amino acid and carbohydrate metabolism, siderophore utilisation, secondary metabolite secretary pathway (Type II and III) and chemotaxis-related genes. The soluble proteome of Δhfq revealed up-regulation of a group of putative lipoproteins by Hfq (Grenga et al. 2017). The role of lipoproteins has been reported in amino acid and carbohydrate metabolism, siderophore utilisation, secondary metabolite secretary and chemotaxis related genes in P. aeruginosa. A genome wide survey predicted lipoprotein PA0953 as thioredoxin, PA2993 and PA1048 to be involved in thiamine biosynthesis and type VI secretion respectively, which is suggestive of the role of lipoprotein in amino acid metabolism, pathogenesis, etc. Furthermore, it was reported that the outer-membrane lipoproteins belonging to the OprM, OmpR, OmpJ encode antibiotic efflux systems; OmpQ for pyoverdine recycling OpmD for quorum sensing (Remans et al. 2010).

To understand the global post-transcriptional effect of Crc on the physiology of P. aeruginosa, transcriptome and proteome of a Crc deficient mutant were analysed against the wild type strain using a post-transcriptional variation (PTV) approach. This study presented a comprehensive map of the Crc post-transcriptional regulon and the mechanism of hierarchical assimilation of carbon sources where Crc played a key role in keeping bacterial homeostasis and consequently metabolic robustness. In addition, the results indicated that CCR in P. aeruginosa is also involved in the regulation of other elements of bacterial physiology such as iron uptake genes, siderophore biosynthesis and uptake, xenosiderophore uptake and heme uptake, although the effect of this regulation on bacterial iron homeostasis has not been explored in detail (Corona et al. 2018).

Many functional studies have shown the mutual cooperation of global posttranscriptional regulators such as Crc and Hfq in controlling the destiny of targeted transcripts. A major loophole in existing understanding has been the lack of structural considerations for cooperation between these global regulators for tight regulation of cellular processes. Pei et al (2018) reported the structural details of Crc using high-resolution cryo-EM structures explaining how Crc can support Hfq in direct translational repression when bound to a translational initiation region on targeted transcripts by forming a multi-component assembly.

RNAseq based transcriptome analysis was carried out with strains of P. aeruginosa PAO1 (wild type), PAO1hfqΔ and PAO1Δcrc. It was found that Hfq-dependent translational regulation was carried out through Crc, Hfq and RNA interaction. This investigation revealed a considerable overlapping interplay between the Crc and Hfq regulon along with protein–protein interaction data supporting rigorous activity between Crc and Hfq protein. Furthermore, the pull-down assays and protein–protein and protein-RNA interactions found that the sRNA binds to the distal surface of Hfq bound Crc complex. In addition to these findings, biochemical and biophysical studies suggest that Crc and Hfq assemble in the presence of RNAs with A-rich motifs (Sonnleitner et al. 2018b). These results were in agreement with the observation of Moreno et al. (2015). The strong interaction between Crc and Hfq enhances the stability of Hfq/Crc/RNA complexes facilitating Hfq-mediated translational repression. Moreover, Crc also interferes with regulatory RNA to Hfq complex formation and thus has a role in riboregulation. In P. aeruginosa hfq deletion strain, it had increased susceptibility to different classes of antibiotics. The transcriptome analyses pointed out the impact of Hfq on mechanisms such as import and efflux, energy metabolism, cell wall composition as well as on the c-di-GMP levels in antibiotic susceptibility. These studies hence have helped in understanding the role of CrcZ in sequestration of Hfq that ultimately enhances the sensitivity to antibiotics (Sonnleitner et al. 2018a).

Conclusion

CCR allows optimum metabolism that is also energetically favorable to achieve efficient growth and enhance the competitiveness of bacteria in their natural habitat. The Crc, Hfq and sRNAs regulate the gene expression in an environmental milieu which has implications in the degradation of various xenobiotic compounds. Complete understanding of the underlying molecular mechanisms behind CCR regulatory networks can help in optimising agricultural, environmental and industrial applications, designing tailor-made biocatalysts and in understanding plant-pathogen or animal-pathogen interactions. The advanced high throughput sequencing and bioinformatics combined with novel approaches including quantitative proteomics, RNAseq and other omics techniques can present a significant breakthrough in discovering and defining exciting mechanisms of regulatory networks.