Drastic Differences in Crh and HPr Synthesis Levels Reflect Their Different Impacts on Catabolite Repression in Bacillus subtilis

In Bacillus subtilis, carbon catabolite repression (CCR) ofcatabolic genes is mediated by ATP-dependent phosphorylationof HPr and Crh. Here we show that the different efficiencieswith which these two proteins contribute to CCR may be due tothe drastic differences in their synthesis rates under conditionsthat cause CCR.

INTRODUCTION

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Introduction

In Bacillus subtilis, carbon catabolite repression (CCR) ofmany catabolic genes is mediated by ATP-dependent phosphorylationof Ser-46 of HPr and of its homologue Crh (4). Although thesetwo proteins exhibit high sequence identity (45%) and are bothefficiently phosphorylated by the HPr kinase/phosphorylase,their contributions to CCR differ (4). P-Ser-Crh can only partlysubstitute for P-Ser-HPr in CCR, whereas P-Ser-HPr can completelysubstitute for P-Ser-Crh in this signal transduction pathway.In order to understand the different behaviors of these twoproteins, we compared the expression levels of the correspondinggenes, crh and ptsH (encoding HPr), in the presence of differentcarbon sources by using transcriptional and translational lacZreporter gene fusions.

MATERIALS AND METHODS

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Materials and Methods

Plasmids, bacterial strains, and growth conditions. The plasmids and the bacterial strains used in this study arelisted in Table 1. Escherichia coli DH5 ${alpha}$ was used as a generalcloning host. Plasmid DNAs of the pMutin4 derivatives were preparedfrom E. coli BMH71-18 (recA⁺) prior to transformation into B.subtilis. E. coli, and B. subtilis strains were routinely grownin Luria-Bertani broth supplemented with the appropriate antibioticswhen necessary (ampicillin at 100 µg/ml for E. coli andchloramphenicol at 5 µg/ml and erythromycin at 0.3 µg/mlfor B. subtilis). Standard procedures were used to transformE. coli (15) and B. subtilis (7). Sequencing of PCR-derivedDNA fragments in the final plasmid constructs was carried outby Genome Express (Meylan, France).

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TABLE 1. Plasmids and bacterial strains

Construction of transcriptional and translational fusions of crh and ptsH to lacZ. To construct the fusion of lacZ to crh-5', the 5' region ofcrh (–120 to +90) was amplified by using the primers BG9(crh [–120 to –103]) and BG10 (crh [+90 to +73]),digested at the HindIII and BamHI sites within the primers,and inserted between these sites in pMutin4, resulting in plasmidpBGM6. To construct the fusion of lacZ to ptsH-5', the 5' regionof ptsH (–276 to +90) was amplified by using the primersLF1 (ptsH [–276 to –258]) and LF2 (ptsH [+90 to+73]), digested at the NotI and HindIII sites within the primers,and inserted between these sites in pMutin4, resulting in plasmidpLF2. Plasmid pBGM8 carrying a ${Phi}$ (crh-lacZ)(Hyb) fusion gene wasconstructed by a three-fragment ligation. The entire crh genewith its ribosome binding site (RBS) was amplified by usingthe primers BG13 (crh [–20 to –3]) and BG14 (crh[+255 to +238]) and digested at the HindIII and XhoI sites withinthe primers. In parallel, the 5' part of lacZ was amplifiedfrom pMutin4 as a template by using the primers BG15 (pMutin2[382 to 399]) and BG16 (pMutin2 [698 to 681]) and digested withXhoI (located within primer BG15) and MstII (occurring naturallywithin lacZ). The two digested PCR fragments were ligated tothe MstII-HindIII vector backbone of pMutin4 (lacking the RBS_spoVGand the lacZ-5' region). A CTC codon (Leu) was inserted togetherwith the XhoI site between the crh and the lacZ parts of thefusion gene, and the Val initiation codon of lacZ had been removed.To obtain the isogenic construct pLF1, which carries a ${Phi}$ (ptsH-lacZ)(Hyb)fusion gene instead of the ${Phi}$ (crh-lacZ)(Hyb) fusion, the ptsHgene was amplified with primers LF3 (ptsH [–20 to –3])and LF4 (ptsH [+264 to +247]), digested at the HindIII and XhoIsites within the primers, and used to replace the HindIII-XhoIfragment (encompassing crh) in pBGM8. The recombinant plasmidsdescribed above were used to transform B. subtilis 168 by asingle crossover event by selection for erythromycin-resistanttransformants. The resulting strains carried the fusions tolacZ described above in the respective chromosomal loci. Foreach strain, the correct structure of the plasmid insertionwas verified by a set of seven different PCRs according to thestrategy published by Pragai and Harwood (13).

Construction of the glcT ${Delta}$ (–16 to +395) deletion mutants. First, the generalized deletion vector pBGM31 was constructedby a three-fragment ligation. The cat gene from plasmid pAC5(9) was isolated as an EcoRI-EheI fragment (1,096 bp). In parallel,a fragment (311 bp) encompassing the three transcriptional terminatorsrrnBt₁t₂- ${lambda}$ t₀ was isolated from pMutin2 by SmaI and PspI digestion.These fragments were ligated to the ClaI-EcoRI vector backboneof pBluescript SK^–. The resulting plasmid, pBGM31, thuscarries a cat-rrnBt₁t₂- ${lambda}$ t₀ cassette encompassed upstream by aSacI-SacII-NotI-SpeI-SmaI-BamHI multiple cloning site and downstreamby a SalI-XhoI-ApaI-KpnI multiple cloning site. Thereafter,the glcT-3' region (+396 to +889) was amplified with primersBG78 (glcT [+396 to +413]) and BG79 (glcT [+889 to +872]), digestedat the BamHI and SacII sites within the primers, and insertedbetween these sites in plasmid pBGM31, resulting in the intermediateplasmid pBGM83. Next, the region upstream of glcT (–599to –17) was amplified with the primers BG76 (glcT [–599to –581]) and BG77 (glcT [–17 to –35]), digestedat the ApaI and XhoI sites within the primers, and insertedbetween these sites in pBGM83. The resulting construct, pBGM84,was linearized by ScaI digestion and used to transform strainsSG57 and SG58 by a double crossover with selection for chloramphenicolresistance. In the resulting strains, SG87 and SG88, the 5'part of glcT (–16 to +395) was deleted and replaced bythe cat-rrnBt₁t₂- ${lambda}$ t₀ cassette reading in the opposite directionof glcT transcription.

Construction of a control strain carrying a Pspac-lacZ cassette inserted in amyE. A control strain (SG68) carrying a constitutively expressedPspac-lacZ cassette on the chromosome was constructed. The Pspacpromoter was amplified with primers BG61 (pMutin2 [143 to 161])and BG23 (pMutin2 [428 to 406]) from pMutin2 (19) as a template,digested with MunI (site located within primer BG61) and BamHI(site located downstream of the Pspac promoter in pMutin2),and inserted between the EcoRI and BamHI sites located in frontof lacZ in pAC6. The resulting plasmid, pBGM55, was linearizedby ScaI digestion and used to transform B. subtilis 168 by adouble crossover with the amyE gene with selection for chloramphenicolresistance.

ß-Galactosidase assays. Overnight cultures of the B. subtilis strains grown in C minimalmedium (9) supplemented with L-tryptophan (100 µg/ml),potassium glutamate (8 g/liter), and one of the various carbonsources (0.5%, wt/vol) as indicated in Tables 2 and 3 were inoculatedat an initial optical density at 600 nm (OD₆₀₀) of 0.15 in thesame medium. IPTG (isopropyl-ß-D-thiogalactopyranoside)(1 mM) was added for induction of the Pspac promoter. The cultureswere grown at 37°C with agitation (160 rpm), and cells wereharvested when the cultures reached an OD₆₀₀ of 0.6 to 0.8.ß-Galactosidase activities were determined as describedby Miller (11), using lysozyme and Triton X-100 treatment (12).The untransformed strain (B. subtilis 168) yielded values ofless than 1 U. Pilot experiments were performed in which theß-galactosidase activities were determined at differenttime points during growth on succinate (lowest growth rate)and glucose (highest growth rate). These experiments revealedthat a steady-state level of ß-galactosidase activitieswas reached with all strains used when the cultures were inthe OD₆₀₀ range of 0.6 to 0.8. In order to test whether growthconditions (i.e., utilization of different carbon sources) maydirectly affect ß-galactosidase activities, we usedthe control strain SG68. The ß-galactosidase activities(in Miller units) obtained for SG68 were as follows: 18 forcitrate, succinate, and gluconate; 23 for myo-inositol; 25 forfructose and glucose; 26 for malate; 27 for sucrose; 28 formannitol; 29 for glucitol; and 30 for glycerol. Thus, the variousgrowth conditions had only a marginal effect on expression oractivity of ß-galactosidase in our assays.

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TABLE 2. ß-Galactosidase activities of strains carrying transcriptional fusions of crh or ptsH to lacZ^a

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TABLE 3. ß-Galactosidase activities of strains carrying translational fusions of crh or ptsH to lacZ^a

RESULTS AND DISCUSSION

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Results and Discussion

In order to compare the transcription rates, we constructedcrh'::lacZ and ptsH'::lacZ transcriptional fusions. The resultingstrains carry an RBS_spoVG-lacZ reporter cassette starting 90bp downstream of the first base of the respective open readingframe (ORF) at their natural chromosomal loci (Fig. 1). In theseconstructs, the complete ORFs of crh and ptsH are restored downstreamof the lacZ cassette insertion sites. Their expression is directedby the IPTG-inducible Pspac promoter present in the insertedcassette in order to overcome polar effects caused by the insertions.The bacteria were grown in minimal medium supplemented withdifferent carbohydrates, and ß-galactosidase activitieswere determined (Table 2). The activities produced by the straincarrying the crh'::lacZ fusion varied only slightly with thecarbon source (29 to 44 U) (Table 2). In contrast, the straincarrying the ptsH'::lacZ fusion showed remarkable differencesin activities. High activities (91 to 268 U) were detectablewhen a substrate of the phosphoenolpyruvate:carbohydrate phosphotransferasesystem (PTS) (mannitol, fructose, sucrose, or glucose) was thecarbon source, whereas significantly lower activities (42 to66 U) were obtained with the other carbohydrates, none of whichis a substrate of the PTS (Table 2). In conclusion, transcriptionof ptsH relative to that of crh was found to be three- to ninefoldstronger when a PTS substrate is utilized (Table 2). Controlexperiments (see Materials and Methods) excluded the possibilitythat the different growth rates may affect the ß-galactosidaseactivities (21).

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FIG. 1. Organization of the crh- and ptsH-containing operons. The gene crh is part of an operon composed of six ORFs which are cotranscribed from a promoter located in front of the gene yvcI (reference 4 and our unpublished data). The gene ptsH can be transcribed from two promoters, one directly in front of ptsH and another in front of the preceding ptsG gene. The hairpin structures correspond to transcriptional terminators. The promoters are indicated by arrows and the corresponding RNAs are indicated by dotted lines, according to previous data (4, 5, 17) and this study. The positions of the transcriptional and translational fusions of lacZ to crh and ptsH, respectively, and of the glcT deletion (–16 to +395) constructed in this study are indicated.

Thereafter, we aimed to compare HPr and Crh synthesis levels.We therefore constructed translational in-frame fusions of theentire crh and ptsH genes to lacZ and placed them at their naturalchromosomal loci preceded by their natural RBSs. The strainwhich carries the chimeric ${Phi}$ (crh-lacZ)(Hyb) fusion gene yieldedß-galactosidase activities that varied only slightlywith the carbon source, with the exceptions of citrate and succinate(Table 3). These substrates caused twofold-higher activitiesthan the other tested substrates. The same effect, althoughless pronounced, was seen with the transcriptional crh'::lacZfusion (Table 2). The strain which carries the ${Phi}$ (ptsH-lacZ)(Hyb)fusion gene produced very high activities (940 to 3,534 U) (Table3). This indicates that ptsH is approximately 10- to 20-foldmore efficiently translated than crh, which is in agreementwith previous observations suggesting that HPr is synthesizedto very large cellular amounts (18). Indeed, the RBS of ptsHis very similar to the optimal B. subtilis RBS (14), whereasthe RBS of crh matches the consensus less well (Fig. 2). Theactivities produced by the ${Phi}$ (ptsH-lacZ)(Hyb) fusion were significantlyhigher when the bacteria were grown on a PTS substrate (1,855to 3,534 U) than when they were grown on a non-PTS carbohydrate(940 to 1,465 U). This confirms our observation that utilizationof a PTS substrate stimulates ptsH transcription (Table 2).The combined effects of stimulated transcription and highlyefficient translation of ptsH amplify the differences in HPrand Crh synthesis levels up to 100-fold (Table 3).

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FIG. 2. Comparison of the RBSs of ptsH and crh. The optimal B. subtilis RBS is as described by Rocha et al. (14).

What may be the mechanism that accounts for the stimulatoryeffect of PTS substrates on ptsH transcription? The genes ptsHand ptsI are cotranscribed from a promoter located directlyin front of ptsH (Fig. 1). This promoter is constitutively activeand not subject to regulation by the carbon source (5). In thepresence of glucose, ptsHI are, in addition, cotranscribed withthe preceding gene ptsG (encoding the glucose-specific enzymeII [EII^Glc]) from a promoter located in front of ptsG (17).Induction of ptsG expression by glucose is mediated by antiterminatorprotein GlcT, which is in turn regulated by (de)phosphorylationvia EII^Glc (2, 16). Upon transport of glucose, EII^Glc dephosphorylatesand activates GlcT, which then prevents formation of a terminatorpresent in the ptsG leader mRNA that otherwise blocks transcriptionelongation (8). This mechanism explains the high ptsH transcriptionrates that we observed in the presence of glucose (Table 2).Furthermore, it was shown that the expression of ptsG is alsoinducible by sucrose (17) and that this up-regulation is mediatedby GlcT (8). To clarify whether cotranscription with ptsG alsoaccounts for the higher ptsH transcription rates in the presenceof various PTS substrates other than glucose, we deleted theleft half of the glcT gene (–16 to +395) in the strainscarrying the transcriptional and translational ptsH-lacZ fusions.This deletion abolishes GlcT antitermination activity but leavesthe ptsG promoter-leader region unaffected (Fig. 1). As canbe seen from the data (Tables 2 and 3), the stimulatory effectof the various PTS substrates was abolished in the ${Delta}$ glcT strains.In contrast, the activities detected in the presence of thevarious non-PTS substrates were not affected by the ${Delta}$ glcT deletion.These results establish (i) that in the absence of PTS substrates,ptsH is expressed exclusively from the promoter directly infront of it, (ii) that additional cotranscription with ptsGcauses higher ptsH transcription rates in the presence of aPTS substrate in general, and (iii) that this cotranscriptionis mediated by antiterminator GlcT.

In conclusion, our data suggest that the highly efficient translationof ptsH and the increased transcription rate in the presenceof PTS carbohydrates, i.e., under conditions of strong CCR,cause a higher cellular concentration of HPr than of Crh. Thesedrastic differences in synthesis level, up to 100-fold, mayexplain why in previous studies (3, 4, 10) only a minor roleof Crh in CCR was detected. Recently it was reported that citM,encoding a Mg²⁺-citrate transporter, is specifically repressedby Crh in a medium composed of glutamate, succinate, and citrate(20). Here we found that the differences in Crh and HPr synthesislevels are lowest (approximately 10-fold) when the bacteriautilize citrate or succinate (Table 3). This observation isin agreement with the proposal (20) for a specific role of Crhunder these conditions.

ACKNOWLEDGMENTS

This research was supported by the CNRS, the Universitéd'Aix-Marseille II, and the Ministère de la Recherche"ACI-jeunes-chercheurs."

We are grateful to F. Denizot for critical reading of the manuscriptand to J. Stülke for gifts of plasmids and for helpfuldiscussion.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Chimie Bactérienne, UPR 9043, Institut de Biologie Structurale et Microbiologie-CNRS, 31 chemin Joseph Aiguier, 13009 Marseille, France. Phone: 33.4.91.16.45.71. Fax: 33.4.91.71.89.14. E-mail: galinier{at}ibsm.cnrs-mrs.fr.

REFERENCES

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