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, 194 (5), 914-24

Output Targets and Transcriptional Regulation by a Cyclic Dimeric GMP-responsive Circuit in the Vibrio Parahaemolyticus Scr Network

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Output Targets and Transcriptional Regulation by a Cyclic Dimeric GMP-responsive Circuit in the Vibrio Parahaemolyticus Scr Network

Rosana B R Ferreira et al. J Bacteriol.

Abstract

The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit.

Figures

Fig 1
Fig 1
New output targets of the Scr circuit. Gene expression was examined in pairs of ΔscrABC and scrABC+ luminescent reporter strains. Strains were spread on multiple HI plates, harvested hourly between 5 and 8 h, and assayed for luminescence. Values reported are the percent activity in the ΔscrABC mutant strain compared to the parental strain for each lux reporter strain at the time corresponding to maximal activity for the parental strain, which was usually 7 h. Strain pairs for each lux fusion (scr mutant/parent) were as follows: VP0829::lux ΔscrC/VP0829::lux (LM9081/LM6321); VPA0267::lux ΔscrABC/VPA0267::lux (LM6565/LM1017); VPA1544::lux ΔscrABC/VPA1544::lux (LM9503/LM8882); VPA0227::lux ΔscrABC/VPA0227::lux (LM9621/LM9376); VPA1598::lux ΔscrABC/VPA1598::lux (LM9492/LM6159); VP1002::lux ΔscrABC/VP1002::lux (LM9497/LM6161); VPA1443::lux ΔscrABC/VPA1443::lux (LM9837/LM6901). The predicted products of these genes are N-acetylglucosamine-6-phosphate deacetylase (VP0829), alkaline serine protease (VPA0227), class 2 lateral flagellar gene product (VPA0267 [FlgE]), class 1 lateral flagellar gene product (VPA1544 [FliR]), N-acetylglucosamine/chitin-binding protein (VPA1598), lipoprotein (VP1002), and type I secretion membrane fusion protein (VPA1443). Error bars indicate standard deviations of the averages of ratios obtained from at least three independent experiments. In all experiments, the reporter activity for each scr mutant was significantly different from the activity in its parental strain (P < 0.0003), with the exception of the control reporter VPA0828::lux pair.
Fig 2
Fig 2
CpsQ affects colony morphology and cpsA expression. (A) IPTG-induced expression of cpsQ induces a crinkly colony morphotype on Congo red medium. Single colonies of LM7794 (wild type/vector) and LM7796 (wild type/cpsQ+) were picked with toothpicks on HI Congo red medium with kanamycin and 0.1 mM IPTG. Colony morphology distinctions were apparent after 2 days growth, continued to develop with room temperature incubation, and were photographed at day 9. (B) IPTG-induced expression of cpsQ increases expression of a cpsA::lacZ reporter fusion. Strains LM7812 (cpsA::lacZ/vector) and LM7814 (cpsA::lacZ/cpsQ+) were spread on HI kanamycin plates with 0.1 mM IPTG. Plates were incubated for 22 h and harvested for β-galactosidase measurements. Error bars indicate standard deviations of triplicate measurements from a representative experiment. The difference was statistically significant (P < 0.0001).
Fig 3
Fig 3
Loss of cpsQ, like cpsR, suppresses the scrA phenotype. cpsA::lacZ expression was assayed in strain LM5984 (wild type) and derivatives LM9690 (ΔscrABC4), LM6241(scrA1), LM6133 (cpsR1), LM9694 (cpsQ2), LM6243 (scrA1 cpsR1), and LM9695 (scrA1 cpsQ2). Cultures were spread on HI plates and incubated for 22 h before harvesting for β-galactosidase assays. Error bars indicate standard deviations from triplicate assays. ***, P < 0.0001.
Fig 4
Fig 4
The mfp operon is regulated by CpsQ. (A) Mutation of cpsQ decreases expression of mfpC::lux. The mfpC::lux reporter strain LM6901 and its cpsQ1::Camr derivative, LM9836, were grown on HI plates and harvested at the indicated times for OD600 and light measurements. Luminescence is reported as normalized light units (SLU). Error bars indicate standard deviations of triplicate light measurements. There is a statistical difference between the wild-type (wt) and cpsQ1 mutant strain at 6 and 7 h (P < 0.002). (B) Organization of the cpsS-cpsQ-mfp locus. Arrows (with gene tag numbers) indicate the direction of transcription and relative size of each gene; the lollipop indicates a predicted transcriptional terminator; sizes of the intergenic regions are indicated in bp. (C) CpsQ activates mfpC::lux in trans. A cosmid carrying the wild-type cpsS-cpsQ-mfp locus and its congenic cpsQ1 derivative were introduced into cpsQ1::Camr mfpC::lux and cpsQ+ mfpC::lux strains. Strains were grown on HI plates with tetracycline and harvested at 7 h for light measurements. Strains included LM10178 (cpsQ1/cpsQ1), L10177 (cpsQ1/cpsQ+), LM10181 (cpsQ+/cpsQ1), and LM10180 (cpsQ+/cpsQ1). Luminescence was measured and is reported as described for panel A. Light production was significantly different for each strain compared to the others (P < 0.0001).
Fig 5
Fig 5
Expression of cpsQ is elevated in scrA mutants and this requires CpsR. A cpsQ::lacZ reporter that was carried on a cosmid was introduced into the following strains: wild type (wt; to make LM9771), ΔscrABC (LM9776), scrA1 (LM9772), cpsR1 (LM9773), and scrA1cpsR1 (LM9774). Cultures were spread on HI tetracycline plates, and the cells were harvested after 22 h of incubation for β-galactosidase measurements. Error bars indicate standard deviations of triplicate measurements. ***, P < 0.001.
Fig 6
Fig 6
cpsQ is epistatic to cpsS, and CpsS regulates cpsR. (A) The cpsA::lacZ allele was recombined onto the chromosome in the indicated backgrounds to make the following reporter strains: wild type (wt; LM5984), ΔcpsS (LM9745), ΔcpsSQ (LM9748), ΔcpsS cpsR1 (LM9039), ΔcpsSQ cpsR1 (LM9041). Expression of cpsA is significantly different in the cpsS mutant compared to the wild type and in the other mutants compared to the cpsS strain (P < 0.001). (B) The cpsR::lacZ reporter carried on a cosmid was introduced into the following backgrounds to make merodiploid reporter strains: wild type (wt; LM10103), ΔcpsS (LM10101), ΔcpsSQ (LM10102). Expression of cpsA was significantly different in two mutant strains compared to the wild type (P < 0.002). Cultures were spread on HI tetracycline plates, and the cells were harvested after 18 h of incubation for β-galactosidase measurements. Error bars indicate standard deviations of triplicate measurements.
Fig 7
Fig 7
CpsQ is sufficient to induce cpsA expression in E. coli. (A) CpsQ stimulates cosmid-derived cpsA::lacZ expression in E. coli. Exponentially growing E. coli strains LLM3727 (DH5α containing the tetracycline-resistant cpsA::lacZ cosmid and the kanamycin resistance expression vector) and LLM3728 (DH5α containing the cpsA::lacZ cosmid and cpsQ+ expression plasmid) were induced in LB medium (with tetracycline, kanamycin, and 0.1 mM IPTG) and grown for 3 h before harvesting for β-galactosidase assays. (B) CpsQ stimulates PcpsA::gfp expression in E coli. The intergenic region upstream of the cpsA coding region was cloned into the pPROBE-gfp vector. Exponentially growing E. coli strains LLM3744 (DH5α containing the ampicillin resistance PcpsA::gfp plasmid and the kanamycin resistance expression vector) and LLM3745 (DH5α containing the PcpsA::gfp plasmid and cpsQ+ expression plasmid) were induced in LB medium (with ampicillin, kanamycin, and 0.1 mM IPTG). Samples were harvested at 3 h for fluorescence and OD600 measurements. Fluorescence is reported as units per ml per OD600 unit. All samples were measured in triplicate (error bars indicate standard deviations). The differences elicited by CpsQ and vector were statistically significant (P < 0.0001 for both panels).
Fig 8
Fig 8
CpsR activates cpsQ expression in E. coli, and CpsQ regulates its own expression. (A) CpsR stimulates cpsQ::lacZ. E. coli strains carrying the cpsQ reporter plasmid and IPTG-inducible expression vector (LLM3975) or cpsR+ clone (LLM3978) were induced using 0.01 mM IPTG and grown for 7 h before harvesting for β-galactosidase assays. LLM3978 produced significantly more activity than LLM3975 (P < 0.0001). (B) CpsR does not stimulate cpsA::lacZ. E. coli strains carrying the cpsA reporter plasmid and the IPTG-inducible expression vector (LLM3727), cpsR+ clone (LLM3982), or cpsQ+ clone (LLM3728) were induced using 0.01 mM IPTG and grown for 6 h before harvesting for β-galactosidase assays. Although the positive-control CpsQ significantly increased cpsA::lacZ expression (P < 0.0001), CpsR did not stimulate the reporter. (C) CpsQ regulates its own expression. E. coli strains carrying the cpsQ reporter plasmid and IPTG-inducible expression vector (LLM3975) or cpsQ+ clone (LLM3977) were induced using 0.1 mM IPTG and grown for 3 h before harvesting for β-galactosidase assays. LLM3977 produced significantly more activity than LLM3975 (P < 0.0001). Exponentially growing strains containing the tetracycline resistance reporter cosmids and the kanamycin resistance expression vector or clones were diluted to an OD600 of 0.1 into LB medium (with tetracycline, kanamycin, and IPTG) and grown for the indicated times before harvesting for β-galactosidase assays. Error bars indicate standard deviations of the mean of triplicate measurements.
Fig 9
Fig 9
CpsQ-dependent regulation of cpsA is dependent on c-di-GMP in E. coli. The Scr circuit was examined in E. coli by introducing components on three plasmids. All strains contained the cpsA::lacZ reporter cosmid (tetracycline resistant). In addition they contained kanamycin resistance plasmids (type 1) encoding cpsQ+ (Q) or the parent vector (V) and ampicillin resistance plasmids (type 2) encoding scrC+ (C), scrABC+ (ABC), or the parent vector (V). These strains included the following: LLM3760 (vector 1 and vector 2), LLM3761 (cpsQ+ and vector 2), LLM3762 (vector 1 and scrC+), LLM3763 (cpsQ+ and scrC+), LLM3752 (cpsQ+ and scrABC+), LLM3757 (vector 1 and scrABC+). The scr and cpsQ genes were IPTG inducible. Strains were grown for 3 h in LB with antibiotics and 0.1 mM IPTG and harvested for β-galactosidase assays (A) and protein samples (B). The SDS-PAGE resolving gel (12%) was stained with Coomassie blue. Lanes: 1, LLM3760; 2, LLM3761; 3, LLM3763; 4, LLM3752; 5, LLM3760.
Fig 10
Fig 10
CpsQ is a c-di-GMP-binding protein. (A) Mutation of a residue in the predicted c-di-GMP-binding pocket produces an altered form of CpsQ (R134A) that fails to activate cpsA::lacZ. Exponentially growing E. coli strains carrying the cpsA::lacZ reporter cosmid and pCOLADuet-1 expression plasmids were induced with 25 or 200 μM IPTG to elicit cpsQ expression. Strains were harvested after 4 h to measure β-galactosidase activity. Strains with the indicated expression plasmid were as follows: LLM3816 (vector), LLM4101 [cpsQ (R134A)], and LLM4102 (cpsQ+). The amount of β-galactosidase in the strain with the His-tagged wild-type CpsQ was significantly different from that in strains with the vector or His-tagged CpsQ (R134A) at both IPTG concentrations (P < 1E−05). (B) Quantification of c-di-GMP released from purified, His-tagged wild-type and mutant CpsQ. Purified CpsQ protein was extracted by two different methods (heat-acid-organic and heat-organic), and the supernatants were examined by liquid chromatography-tandem mass spectrometry. Each bar represents the average of 2 independent extractions (error bars indicate standard deviations), with the exception of heat-organic extraction for Q (R134A); due to limiting amounts of protein, only one extraction of this type was performed (thus, there is no error bar). Two batches of wild-type CpsQ were purified and extracted (WT Q-1 and WT Q-2 from LLM3829 and LLM4095, respectively) and one of the mutant (Q R134A) from LLM4093. Statistical differences between the wild type and mutant were significant (P < 0.001, for comparison of the combined WT Q-1 or Q-2 extractions with the combined mutant extractions).
Fig 11
Fig 11
Model for the Scr network controlling swarming and sticking. ScrC modulates the level of c-di-GMP, and its activity is controlled by the influence of ScrA and ScrB. ScrA produces a cell-cell signaling molecule, the S-signal, which upon accumulation elicits ScrB-dependent stimulation of the phosphodiesterase activity of ScrC. Other sensors, such as ScrG, can also feed into this circuit to impact c-di-GMP. A high level of c-di-GMP promotes production of capsular polysaccharide production and biofilm formation, while a low concentration of this signaling molecule favors swarming motility. New output targets identified in this work are listed in Table S2 of the supplemental material. They include c-di-GMP-repressible members of the surface-sensing regulon, including genes encoding a protease, GpbA (a cell surface adhesin predicted to bind chitin or N-acetylglucosamine), and the type III secretion system on chromosome 1 (T3SS1). They also include c-di-GMP-inducible genes, such as the mfp operon, which encodes a type I membrane fusion transport system and its putative secreted calcium-binding substrate, that plays a role in biofilm development. Although how c-di-GMP represses the surface-sensing regulon remains to be elucidated (indicated by the question mark), elements of the positive regulatory circuit governing expression of cps and mfp expression are defined in this work. The circuit involves three transcriptional regulators. CpsQ is the direct regulator of expression of cps biosynthetic genes; it also regulates its own expression. CpsQ binds c-di-GMP. This second messenger influences the activity and stability of CpsQ. CpsR positively regulates cpsQ, and CpsS prevents expression of cpsR, although it is not known whether it works directly (indicated by dashed line).

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