Carbon Storage Regulator Research Articles

Damage to the microbial cell membrane during pyrolytic sugar utilization and strategies for increasing resistance.

Lignocellulosic biomass is an appealing feedstock for the production of biorenewable fuels and chemicals, and thermochemical processing is a promising method for depolymerizing it into sugars. However, trace compounds in this pyrolytic sugar syrup are inhibitory to microbial biocatalysts. This study demonstrates that hydrophobic inhibitors damage the cell membrane of ethanologenic Escherichia coli KO11+lgk. Adaptive evolution was employed to identify design strategies for improving pyrolytic sugar tolerance and utilization. Characterization of the resulting evolved strain indicates that increased resistance to the membrane-damaging effects of the pyrolytic sugars can be attributed to a glutamine to leucine mutation at position 29 of carbon storage regulator CsrA. This single amino acid change is sufficient for decreasing EPS protein production and increasing membrane integrity when exposed to pyrolytic sugars.

Conflicting objectives in production forests pose a challenge for forest management

Conflicts among different ecosystem services have been shown to be common and potentially exacerbated by management interventions. In order to improve the sustainability of natural resource use, the occurrence of these conflicts and the effects that management actions have on them need to be understood. We studied the conflicts between ecosystem services and the potential to solve them by management choices in boreal production forests. Our study area consisted of nearly 30,000 forest stands which were simulated for 50years into the future under alternative management scenarios. The study included four ecosystem services – timber production, bilberry production, carbon storage, and pest regulation – and one biodiversity conservation objective defined as availability of deadwood resources. We 1) measured the conflicts among each pair of objectives, and 2) identified a compromise solution for each pairwise conflict defined as one which simultaneously minimizes the losses for both objectives. Our results show that conflicts between timber production and other objectives are typical, severe, and difficult to solve, while non-extractive benefits including biodiversity conservation can be more easily reconciled with each other. To mitigate the most severe conflicts in boreal forests, increased diversity in management regimes is required.

Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus.

Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and in vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocagin A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.

Indole enhances the survival of Pantoea ananatis YJ76 in face of starvation conditions.

Pantoea ananatis YJ76 is an indole-producing predominant diazotrophic endophyte isolated from rice having multiple growth-promoting effects on host plant. As a decomposition metabolite of L-tryptophan (L-Trp), indole is confirmed to regulate various physiological processes of bacteria. In this research, we found that indole significantly improves the survival of YJ76 in face of starvation conditions and the promoting effect is related to the glycogen accumulation promoted by indole, which is much more significant in the middle decline phase than in other growth phases. Since carbon storage regulator CsrA is a key inhibiting factor on the storage of glycogen in bacteria, we explored the relation between indole-enhanced glycogen accumulation and csrA expression and found that there is a positive correlation between indole-enhanced glycogen accumulation and the indole-inhibited csrA expression in YJ76, which implies the potential relation between CsrA regulation and indole regulatory pathway.

The Legionella pneumophila genome evolved to accommodate multiple regulatory mechanisms controlled by the CsrA-system.

The carbon storage regulator protein CsrA regulates cellular processes post-transcriptionally by binding to target-RNAs altering translation efficiency and/or their stability. Here we identified and analyzed the direct targets of CsrA in the human pathogen Legionella pneumophila. Genome wide transcriptome, proteome and RNA co-immunoprecipitation followed by deep sequencing of a wild type and a csrA mutant strain identified 479 RNAs with potential CsrA interaction sites located in the untranslated and/or coding regions of mRNAs or of known non-coding sRNAs. Further analyses revealed that CsrA exhibits a dual regulatory role in virulence as it affects the expression of the regulators FleQ, LqsR, LetE and RpoS but it also directly regulates the timely expression of over 40 Dot/Icm substrates. CsrA controls its own expression and the stringent response through a regulatory feedback loop as evidenced by its binding to RelA-mRNA and links it to quorum sensing and motility. CsrA is a central player in the carbon, amino acid, fatty acid metabolism and energy transfer and directly affects the biosynthesis of cofactors, vitamins and secondary metabolites. We describe the first L. pneumophila riboswitch, a thiamine pyrophosphate riboswitch whose regulatory impact is fine-tuned by CsrA, and identified a unique regulatory mode of CsrA, the active stabilization of RNA anti-terminator conformations inside a coding sequence preventing Rho-dependent termination of the gap operon through transcriptional polarity effects. This allows L. pneumophila to regulate the pentose phosphate pathway and the glycolysis combined or individually although they share genes in a single operon. Thus the L. pneumophila genome has evolved to acclimate at least five different modes of regulation by CsrA giving it a truly unique position in its life cycle.

Tissue dual RNA-seq allows fast discovery of infection-specific functions and riboregulators shaping host–pathogen transcriptomes

Pathogenic bacteria need to rapidly adjust their virulence and fitness program to prevent eradication by the host. So far, underlying adaptation processes that drive pathogenesis have mostly been studied in vitro, neglecting the true complexity of host-induced stimuli acting on the invading pathogen. In this study, we developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens. Using this tool for Yersinia pseudotuberculosis-infected lymphatic tissues, we revealed numerous alterations of host transcripts associated with inflammatory and acute-phase responses, coagulative activities, and transition metal ion sequestration, highlighting that the immune response is dominated by infiltrating neutrophils and elicits a mixed TH17/TH1 response. In consequence, the pathogen's response is mainly directed to prevent phagocytic attacks. Yersinia up-regulates the gene and expression dose of the antiphagocytic type III secretion system (T3SS) and induces functions counteracting neutrophil-induced ion deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this response. The strongest influence on virulence was found for the loss of the carbon storage regulator (Csr) system, which is shown to be essential for the up-regulation of the T3SS on host cell contact. In summary, our established approach provides a powerful tool for the discovery of infection-specific stimuli, induced host and pathogen responses, and underlying regulatory processes.

Impacts of conversion of tropical peat swamp forest to oil palm plantation on peat organic chemistry, physical properties and carbon stocks

Ecosystem services provided by tropical peat swamp forests, such as carbon (C) storage and water regulation, are under threat due to encroachment and replacement of these natural forests by drainage-based agriculture, commonly oil palm plantation. This study aims to quantify how the chemical and physical properties of peat change during land conversion to oil palm. This will be addressed by comparing four separate stages of conversion; namely, secondary peat swamp forests, recently deeply drained secondary forests, cleared and recently planted oil palm, and mature oil palm plantation in North Selangor, Malaysia. Results indicate accelerated peat decomposition in surface peats of mature oil palm plantations due to the lowered water table and altered litter inputs associated with this land-use change. Surface organic matter content and peat C stocks at secondary forest sites were higher than at mature oil palm sites (e.g. C stocks were 975±151 and 497±157Mgha−1 at secondary forest and mature oil palm sites, respectively). Land conversion altered peat physical properties such as shear strength, bulk density and porosity, with mirrored changes above and below the water table. Our findings suggest close links between the organic matter and C content and peat physical properties through the entire depth of the peat profile. We have demonstrated that conversion from secondary peat swamp forest to mature oil palm plantation may seriously compromise C storage and, through its impact on peat physical properties, the water holding capacity in these peatlands.

Structural basis for the CsrA-dependent modulation of translation initiation by an ancient regulatory protein

Regulation of translation is critical for maintaining cellular protein levels, and thus protein homeostasis. The conserved RNA-binding protein CsrA (also called RsmA; for carbon storage regulator and regulator of secondary metabolism, respectively; hereafter called CsrA) represents a well-characterized example of regulation at the level of translation initiation in bacteria. Binding of a CsrA homodimer to the 5'UTR of an mRNA occludes the Shine-Dalgarno sequence, blocking ribosome access for translation. Small noncoding RNAs (sRNAs) can competitively antagonize CsrA activity by a well-understood mechanism. However, the regulation of CsrA by the protein FliW is just emerging. FliW antagonizes the CsrA-dependent repression of translation of the flagellar filament protein, flagellin. Crystal structures of the FliW monomer reveal a novel, minimal β-barrel-like fold. Structural analysis of the CsrA/FliW heterotetramer shows that FliW interacts with a C-terminal extension of CsrA. In contrast to the competitive regulation of CsrA by sRNAs, FliW allosterically antagonizes CsrA in a noncompetitive manner by excluding the 5'UTR from the CsrA-RNA binding site. Our phylogenetic analysis shows that the FliW-mediated regulation of CsrA regulation is the ancestral state in flagellated bacteria. We thus demonstrate fundamental mechanistic differences in the regulation of CsrA by sRNA in comparison with an ancient regulatory protein.

FliW antagonizes CsrA RNA binding by a noncompetitive allosteric mechanism

CsrA (carbon storage regulator A) is a widely distributed bacterial RNA binding protein that regulates translation initiation and mRNA stability of target transcripts. In γ-proteobacteria, CsrA activity is competitively antagonized by one or more small RNAs (sRNAs) containing multiple CsrA binding sites, but CsrA in bacteria outside the γ-proteobacteria is antagonized by a protein called FliW. Here we show that FliW of Bacillus subtilis does not bind to the same residues of CsrA required for RNA binding. Instead, CsrA mutants resistant to FliW antagonism (crw) altered residues of CsrA on an allosteric surface of previously unattributed function. Some crw mutants abolished CsrA-FliW binding, but others did not, suggesting that FliW and RNA interaction is not mutually exclusive. We conclude that FliW inhibits CsrA by a noncompetitive mechanism that differs dramatically from the well-established sRNA inhibitors. FliW is highly conserved with CsrA in bacteria, appears to be the ancestral form of CsrA regulation, and represents a widespread noncompetitive mechanism of CsrA control.

Genome engineering Escherichia coli for L-DOPA overproduction from glucose.

Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose.

On the Kinetic and Allosteric Regulatory Properties of the ADP-Glucose Pyrophosphorylase from Rhodococcus jostii: An Approach to Evaluate Glycogen Metabolism in Oleaginous Bacteria

Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.

Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins

The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIAGlc of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3′ segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source.

Genetic Manipulation of Glycogen Allocation Affects Replicative Lifespan in E. coli.

In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported.

The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated. Here, a multiscale analysis was performed in a wild-type strain and its isogenic mutant attenuated for CsrA (including growth parameters, gene expression levels, metabolite pools, abundance of enzymes and fluxes). Data integration and regulation analysis showed a coordinated control of the expression of glycolytic enzymes. This also revealed the imbalance of metabolite pools in the csrA mutant upper glycolysis, before the phosphofructokinase PfkA step. This imbalance is associated with a glucose-phosphate stress. Restoring PfkA activity in the csrA mutant strain suppressed this stress and increased the mutant growth rate on glucose. Thus, the carbon storage regulator system is essential for the effective functioning of the upper glycolysis mainly through its control of PfkA. This work demonstrates the pivotal role of post-transcriptional regulation to shape the carbon metabolism.

Soil Functional Zone Management: A Vehicle for Enhancing Production and Soil Ecosystem Services in Row-Crop Agroecosystems.

There is increasing global demand for food, bioenergy feedstocks and a wide variety of bio-based products. In response, agriculture has advanced production, but is increasingly depleting soil regulating and supporting ecosystem services. New production systems have emerged, such as no-tillage, that can enhance soil services but may limit yields. Moving forward, agricultural systems must reduce trade-offs between production and soil services. Soil functional zone management (SFZM) is a novel strategy for developing sustainable production systems that attempts to integrate the benefits of conventional, intensive agriculture, and no-tillage. SFZM creates distinct functional zones within crop row and inter-row spaces. By incorporating decimeter-scale spatial and temporal heterogeneity, SFZM attempts to foster greater soil biodiversity and integrate complementary soil processes at the sub-field level. Such integration maximizes soil services by creating zones of ‘active turnover’, optimized for crop growth and yield (provisioning services); and adjacent zones of ‘soil building’, that promote soil structure development, carbon storage, and moisture regulation (regulating and supporting services). These zones allow SFZM to secure existing agricultural productivity while avoiding or minimizing trade-offs with soil ecosystem services. Moreover, the specific properties of SFZM may enable sustainable increases in provisioning services via temporal intensification (expanding the portion of the year during which harvestable crops are grown). We present a conceptual model of ‘virtuous cycles’, illustrating how increases in crop yields within SFZM systems could create self-reinforcing feedback processes with desirable effects, including mitigation of trade-offs between yield maximization and soil ecosystem services. Through the creation of functionally distinct but interacting zones, SFZM may provide a vehicle for optimizing the delivery of multiple goods and services in agricultural systems, allowing sustainable temporal intensification while protecting and enhancing soil functioning.

Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

The global regulatory system Csr senses glucose through the phosphoenolpyruvate: carbohydrate phosphotransferase system.

A novel connection between two regulatory systems controlling crucial biological processes in bacteria, the carbon storage regulator (Csr) system and the glucose-specific phosphotransferase system (PTS), is reported by Leng et al. in this issue. This involves the interaction of unphosphorylated EIIA(Glc), a component of the glucose-specific PTS, with the CsrD protein, which accelerates the decay of the CsrB and CsrC small RNAs via RNase E in Escherichia coli. As unphosphorylated EIIA(G) (lc) is generated in the presence of glucose, the PTS thus acts as a sensor of glucose for the Csr system. Interestingly, another pathway can operate for communication between the Csr system and the glucose-specific PTS. The absence of glucose generates phosphorylated EIIA(Glc) , which activates the enzyme adenylate cyclase to produce cyclic adenosine monophosphate (cAMP) that, in turn, binds to the regulator cAMP receptor protein (CRP). Leng et al. show that the complex cAMP-CRP modestly reduces CsrB decay independently of CsrD. On the other hand, a previous study indicates that the complex cAMP-CRP positively regulates the transcription of CsrB and CsrC in Salmonella enterica. Therefore, EIIA(G) (lc) could work as a molecular switch that regulates the activity of the Csr system, in response to its phosphorylation state determined by the presence or absence of glucose, in order to control gene expression.

Abundant Intergenic TAACTGA Direct Repeats and Putative Alternate RNA Polymerase β' Subunits in Marine Beggiatoaceae Genomes: Possible Regulatory Roles and Origins.

The genome sequences of several giant marine sulfur-oxidizing bacteria present evidence of a possible post-transcriptional regulatory network that may have been transmitted to or from two distantly related bacteria lineages. The draft genome of a Cand. “Maribeggiatoa” filament from the Guaymas Basin (Gulf of California, Mexico) seafloor contains 169 sets of TAACTGA direct repeats and one indirect repeat, with two to six copies per set. Related heptamers are rarely or never found as direct repeats. TAACTGA direct repeats are also found in some other Beggiatoaceae, Thiocystis violascens, a range of Cyanobacteria, and five Bacteroidetes. This phylogenetic distribution suggests they may have been transmitted horizontally, but no mechanism is evident. There is no correlation between total TAACTGA occurrences and repeats per genome. In most species the repeat units are relatively short, but longer arrays of up to 43 copies are found in several Bacteroidetes and Cyanobacteria. The majority of TAACTGA repeats in the Cand. “Maribeggiatoa” Orange Guaymas (BOGUAY) genome are within several nucleotides upstream of a putative start codon, suggesting they may be binding sites for a post-transcriptional regulator. Candidates include members of the ribosomal protein S1, Csp (cold shock protein), and Csr (carbon storage regulator) families. No pattern was evident in the predicted functions of the open reading frames (ORFs) downstream of repeats, but some encode presumably essential products such as ribosomal proteins. Among these is an ORF encoding a possible alternate or modified RNA polymerase beta prime subunit, predicted to have the expected subunit interaction domains but lacking most catalytic residues. A similar ORF was found in the Thioploca ingrica draft genome, but in no others. In both species they are immediately upstream of putative sensor kinase genes with nearly identical domain structures. In the marine Beggiatoaceae, a role for the TAACTGA repeats in translational regulation is suggested. More speculatively, the putative alternate RNA polymerase subunit could be a negative transcriptional regulator.

Metabolic engineering of Escherichia coli for the production of phenylalanine and related compounds

In this review, the metabolic engineering approaches including those used by the authors in creating phenylalanine producers based on Escherichia coli were systematized. Optimization of the amino acid biosynthesis was conducted in order to obtain significant quantities of phenylalanine and to ensure the availability of its direct precursors in cell metabolism—erythrose 4-phosphate and phosphoenolpyruvic acid. The possibility of altering global regulation mechanisms was investigated for a full reorientation of the metabolism to phenylalanine synthesis with the use of Csr (carbon storage regulator). The identification of the aromatic amino acids exporter YddG is associated with the use of the phenylalanine producer as a test-system. Novel approaches to phenylalanine producer construction (use of the “leaky” allele tyrA—ssrA, promoters of the phosphate regulon), as well as new methods of obtaining producers of similar amino acid tyrosine, were discussed. Examples of the synthesis of useful aromatic compounds from phenylalanine or its precursor, phenylpyruvic acid, with E. coli as the ecipient for foreign gene expression were examined.

CRP-Mediated Carbon Catabolite Regulation of Yersinia pestis Biofilm Formation Is Enhanced by the Carbon Storage Regulator Protein, CsrA.

The natural transmission of Yersinia pestis is reliant upon biofilm blockage of the flea vector. However, the environmentally-responsive adaptive regulators which facilitate Y. pestis biofilm production in accordance with the flea midgut milieu are not well understood. We seek to establish the impact of available carbon source metabolism and storage upon Y. pestis biofilm production. Our findings demonstrate that Y. pestis biofilm production is subject to carbon catabolite regulation in which the presence of glucose impairs biofilm production; whereas, the sole metabolism of alternate carbon sources promotes robust biofilm formation. This observation is facilitated by the cAMP receptor protein, CRP. In accordance with a stark growth defect, deletion of crp in both CO92 and KIM6+ Y. pestis strains significantly impaired biofilm production when solely utilizing alternate carbon sources. Media supplementation with cAMP, a small-molecule activator of CRP, did not significantly alter Y. pestis biofilm production. Furthermore, CRP did not alter mRNA abundance of previously-characterized hms biofilm synthesis and regulation factors. Therefore, our findings indicate CRP does not confer a direct stimulatory effect, but may indirectly promote Y. pestis biofilm production by facilitating the alternate carbon source expression profile. Additionally, we assessed the impact of the carbon storage regulator protein, CsrA, upon Y. pestis biofilm production. Contrary to what has been described for E. coli, Y. pestis biofilm formation was found to be enhanced by CsrA. Regardless of media composition and available carbon source, deletion of csrA significantly impaired Y. pestis biofilm production. CsrA was found to promote Y. pestis biofilm production independent of glycogen regulation. Loss of csrA did not significantly alter relative hmsH, hmsP, or hmsT mRNA abundance. However, deletion of hmsP in the csrA-deficient mutant enabled excessive biofilm production, suggesting CsrA enables potent Y. pestis biofilm production through cyclic diguanylate regulation.