JOURNAL

OF

BACTERIOLOGY, Jan. 1991, p. 783-790

Vol. 173,

0021-9193/91/020783-08$02.00/0 Copyright © 1991, American Society for Microbiology

No. 2

Regulation of Proline Utilization in Enteric Bacteria: Cloning and Characterization of the Klebsiella put Control Region LI-MEI CHEN

AND

STANLEY MALOY*

Department of Microbiology, University of Illinois, 131 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 60801 Received 16 July 1990/Accepted 7 November 1990

Enteric bacteria can grow on proline as the sole nitrogen and carbon source. Expression of the proline utilization (put) operon in Klebsiella strains and Escherichia coli is responsive to nitrogen regulation. In contrast, Salmonella typhimurium cannot activate put operon expression when growing in medium with glucose as a carbon source and proline as the sole nitrogen source. To compare nitrogen regulatory sites in the control regions of the put operons in these three closely related genera, we cloned the Klebsiella put operon onto a plasmid. The putA and putP genes were localized on the plasmid by transposon mutagenesis. The DNA sequence of the put control region was determined and compared with those of the put control regions from S. typhimurium and E. coli. The overall size and organization of the put control region were very similar in all three bacteria. However, no obvious ntr regulatory sites were found in this region, and transcription of the put genes started at the same sites during growth with limiting or excess nitrogen. These results strongly suggested that the Klebsiella put operon may not be directly regulated by the ntr system.

Many enteric bacteria can use proline as the sole carbon and nitrogen source. The genes for proline utilization (put) are located at 22 min on the genetic maps of Escherichia coli (5) and Salmonella typhimurium (38). The putP gene encodes the primary proline permease, an integral membrane protein that catalyzes proline-sodium symport (25). After uptake, proline is degraded to glutamate by the putA gene product, a bifunctional enzyme with both proline oxidase and pyrroline-5-carboxylic acid dehydrogenase activities

teric bacteria (22).

Nitrogen regulation requires at least three ntrA, ntrB, and ntrC. The ntrA gene encodes a sigma factor (u54) that confers the specificity of the RNA polymerase holoenzyme for nitrogen-regulated promoters. The ntrB gene product is a phosphorylase that dephosphorylates the NtrC protein when excess nitrogen is available. The phosphorylated NtrC protein is an activator that binds gene products:

to "enhancer sites" and catalyzes isomerization of the closed a54-RNA polymerase-promoter complex to an open complex (34). The consensus sequences for the NtrC-binding site and the NtrA promoter have been identified (22). The DNA sequences of the put control regions from E. coli and S. typhimurium have been determined. To determine the molecular basis for the difference in the nitrogen regulation of the put operons in these three closely related genera, we cloned the Klebsiella put operon and determined the DNA sequence of the put control region and the transcriptional start sites used during growth under conditions of nitrogen limitation and nitrogen excess. The DNA sequence of the Klebsiella put control region was searched for regulatory sites and compared with those of the put control regions of E. coli and S. typhimurium.

(27).

Expression of the put operon is regulated by proline. Genetic and molecular studies in S. typhimurium have indicated that the PutA protein also represses transcription of the put operon in the absence of proline (28, 32). In addition to being induced by proline, put operon expression is catabolite repressed: cyclic AMP receptor protein and cyclic AMP are required for full expression of the put operon (19, 26). Since proline can serve as the sole nitrogen source, the put operon is expected to be nitrogen regulated. Previous studies showed that the catabolite repression of the Klebsiella put operon and the E. coli put operon is relieved during nitrogen starvation (33, 35). In contrast, nitrogen starvation does not efficiently relieve catabolite repression of the S. typhimurium put operon, so S. typhimurium is unable to grow in medium with glucose as a carbon source and proline as the sole nitrogen source (35). There seems to be a hierarchy of nitrogen regulation of the put operon in these three closely related enteric bacteria: the Klebsiella put operon is induced to high levels during nitrogen starvation, the E. coli put operon is induced to intermediate levels during nitrogen starvation, and the S. typhimurium put operon is not induced by nitrogen starvation. These differences could arise from mutations in nitrogen regulatory sites in the put regulatory region or physiological differences in the nitrogen regulatory systems of the three bacteria. Nitrogen regulation has been extensively studied in en*

MATERIALS AND METHODS Klebsiella nomenclature. The Klebsiella strains used in this study are all derivatives of KC1043, a phage P1-sensitive mutant of strain W-70 which has been cured of endogenous plasmids (6). These strains have been frequently classified as Klebsiella aerogenes (39); however, they seem to be closely related to strains previously classified as K. aerogenes 1033 and now commonly designated K. pneumoniae. Although the name K. pneumoniae is officially correct (23), there is still considerable confusion about the nomenclature and relatedness of commonly used Klebsiella strains. Construction of strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. P1 transductions and matings were done as described by Miller (29). P22 transductions were done as described by Maloy

Corresponding author.

(24). 783

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TABLE 1. Bacterial strains and plasmids

Strains S. typhimurium TR2619 TT2272 TR5878 MS1995 E. coli RM2 JA200

LCD63 EM450 EM451 Klebsiella sp. KC1043 KC1467

Source

Relevant genotype or phenotype

Strain or plasmid

A(putPA)516a A(putPA)524 zcc-7::TnlO hsdLT6 hsdS29 galE550 TR5878 A(putPA)524 zcc-7::TnJO

A(putPA)JO1 recA thr leu AtrpES lacY (F+) hsdR metB A(srl::TnlO-recA) JA200(pKC7) RM2 A(srl::TnJO-recA)

hutCSS KC1043 Mu cts62 HP1 (pEG5005)

Ratzkin and Roth (36) Menzel and Roth (27) J. Roth This study J. Wood (41) J. Cronan Hahn et al. (18) This study This study R. Bender R. Bender

Plasmidsb pEG5005

Groisman and Casadaban (17) Mu cts Kanr rep (pMB1) This study Kanr putP+ putA+ Mu cts 62 A' B+ pKM10 This study Kanr putP+ putA+ Mu cts62 A B (deletion of DraI [0.93]-Dral [3.42] on pKM10) pKC7 This study Ampr (subcloned pKC7 HindIII [12.5]-HindIII [12.9] in pTZ18R HindlIl) pKC9 This study Kanr put Mu cts62 A B (deletion of BamHI [5.3]-BamHI [14.0] on pKC7) pKC14 a This is a large deletion which removes the entire put operon and flanking sequences. The selection is nontransducible by phage P22 (>50 kb). b

The numbers in brackets indicate the map coordinants of the restriction sites in kilobases. Derivatives of pKC7 with TnlOOO insertions are shown in Fig.2

EM450 was constructed by transforming plasmid pKC7 into JA200. EM451 was constructed by transduction of RM2 with a P1 phage lysate grown on LCD63, selection for Tet', and scoring for UV sensitivity to confirm that the recA mutation was coinherited with TnJO. MS1995 was constructed by transduction of TR5878 with a P22 phage lysate grown on TT2272 (AputPAS24 zcc-7::TnJO), selection for Tetr, and screening for Put- transductants. Media and growth conditions. Nutrient broth (NB) (Difco) supplemented with 0.5% NaCl was used for rich medium. Three types of minimal media were used. E medium (40) and NCE medium (36) were supplemented with 0.6% succinate or 0.2% glucose as a carbon source. Both E medium and NCE medium contained NH4+ as a nitrogen source. When growth on proline as a sole nitrogen source was selected, NCN medium (40 mM KH2PO4, 80 mM K2HPO4) with 0.2% proline and 0.6% succinate was used (PSN). Proline-TTC (2,3,5-triphenyl tetrazolium chloride) indicator plates (27) were used to screen for Put+ clones. To select for plasmids, we added ampicillin to rich medium at 70 p.g/ml and kanamycin to rich medium at 50 ,ug/ml and to minimal media at 250 ,ug/ml. When needed to supplement auxotrophic requirements, vitamins and amino acids were added to minimal media at the final concentrations described by Davis et al. (11). Cloning of the KlebsieUa put operon. Cloning of the Klebsiella put operon from KC1467 was done as described by Groisman and Casadaban (17). To select for Put+ clones, we grew the recipient strain MS1995 overnight in NB with 1% glucose, pelleted it, and resuspended it in an equal volume of 5 mM MgSO4-5 mM CaCl2. An equal volume of the Mu phage lysate was mixed with the MS1995 culture; the mixture was incubated at 30°C for 4 h to allow phenotypic expression of Kanr, spread on NB-kanamycin plates, and

incubated at 30°C for 2 days. The transductants were replica plated onto PSN plates to select for Ptit' clones. Mutagenesis of plasmid DNA with Tn1000. TnJ000 insertions in the cloned put genes were isolated essentially as described by Maloy (24). The donor strain, EM450, was grown in 2 ml of NB-kanamycin to the mid-log phase and mixed with an equal volume of an overnight culture of the recipient strain, EM451. After incubation at 37°C for 4 h, the mixture was centrifuged and the cell pellet was spread on E medium-glucose-kanamycin plates supplemented with tryptophan and thiamine. The plates were incubated at 37°C for 2 days, and the Kanr colonies were replica plated onto proline-TTC plates and screened for Put- mutants. Expression of plasmid proteins. Plasmids carrying the Klebsiella put' operon or put: :TnlOOO insertions were transformed into E. coli LCD63. Maxicell analysis was done as described by Hahn et al. (18) with minor modifications. During the labeling, 0.5 mM phenylmethylsulfonyl fluoride was added to the reaction mixture to decrease proteolytic degradation of truncated peptides produced by the put:: TnJOOO mutants. S30 extracts for in vitro transcriptiontranslation coupling reactions were purchased from Amersham Corp. Reactions were performed in accordance with the supplier's specifications.Primer extension analysis. Transcriptional start sites of putA and putP genes were determined by primer extension analysis. Total bacterial RNA was isolated by a hot sodium dodecyl sulfate-urea method (2). End-labeling reactions and primer extension analysis were done essentially as described by Aubusel et al. (4) with minor modifications: end-labeled primers were separated from unincorporated [32P]ATP on a Sep-Pak column (37), and 1 p.l of actinomycin D (4 mg/ml in 80% ethanol) was added to each sample during reverse

VOL. 173, 1991

REGULATION OF PROLINE UTILIZATION IN ENTERIC BACTERIA

transcription. Transcripts were analyzed by electrophoresis in 8% polyacrylamide denaturing gels. Molecular techniques. Restriction enzymes and T4 DNA ligase were obtained from Bethesda Research Laboratories, Inc. DNA ligation and restriction digests were performed in accordance with the enzyme supplier's specifications. Plasmid DNA was isolated by an alkaline lysis method and separated by electrophoresis in 0.8% agarose gels in TBE buffer (37). Transformation with plasmid DNA was done by the polyethylene glycol-dimethyl sulfoxide procedure (9) or electroporation (32). Dideoxy sequencing was done with modified T7 polymerase (United States Biochemicals, Cleveland, Ohio). Two strategies were used for double strand sequencing of the Klebsiella put clones. Plasmid pKC9 was constructed by subcloning pKC7 HindIII fragments onto pTZ18, and the universal primer was used to sequence from the HindIll site into the regulatory region. In addition, pKC7 derivatives carrying Tn1000 insertions near the put regulatory region were digested with restriction enzymes that cut within TnlOO0 and within the cloned put fragment. DNA fragments containing only the right or left end of Tn1000 were subcloned into pTZ18. A synthetic oligonucleotide complementary to the inverted repeats at each end of Tn1000 (10) was used as a primer for sequencing toward the put regulatory region. Proline permease and proline oxidase activities. Proline permease activity was measured by determining the sensitivity to the toxic proline analogs dehydroproline (DHP) and azetidine-2-carboxylic acid (AZT) as described by Hahn and Maloy (19) or by directly assaying proline transport as described by Ekena et al. (14). Proline oxidase activity was assayed as described by Dendinger and Brill (13).

RESULTS Cloning of the Klebsiella put operon. The Klebsiella put operon was cloned onto Mud5005 by an in vivo cloning procedure (17). An S. typhimurium strain with a nontransducible put deletion was infected with a random collection of Mud5005 clones, selecting for Kanr. The Kanr colonies were replica plated onto proline-TTC plates to identify potential Put' clones. Five Put' clones were obtained from 7,000 Kanr transductants. The sizes of the plasmids carrying the put genes ranged from 18 to 35 kb, but all of the plasmids carried a common region, as determined on the basis of the similarity of their restriction maps. A restriction map of plasmid pKM10 is shown in Fig. 1A. Since these plasmids carry a functional Mu transposase, they are prone to rearrangements when moved into new strains because of zygotic induction. Therefore, a derivative of pKM10 with a partial deletion of the Mu transposase (pKC7) was used for further studies (Fig. 1B). Expression of the put operon in this clone was confirmed by measuring proline transport and proline oxidase activities (Table 2). The results indicated that pKC7 carried both the putP gene and the putA gene. Defining the put operon. To locate the putA and putP structural genes, we isolated random Tn1000 insertions in the cloned put operon. The restriction sites on the plasmids containing put::Tn1000 insertion mutations were mapped to determine the locations of the TnlOOO insertions. On the basis of the positions of the TnJOOO insertion mutations, the put operon was localized to a 5.2-kb region on pKC7 (Fig. 2). The locations of the putA and putP genes on the physical map were determined by measuring the analog sensitivity of the put::TnJOOO mutants. DHP and AZT are proline analogs which can be transported into cells by the PutP protein.

785

When transported into cells, both analogs are incorporated into proteins and inhibit cell growth (15). At the concentrations used in this study, EM451 (Z\putPA) is resistant to AZT (because the putP mutation prevents AZT transport) and sensitive to DHP (because a small amount of DHP is transported into the cell by the proP gene product but cannot be degraded in the absence of the PutA protein). In contrast, pKC7 complements the chromosomal Aput mutation, resulting in sensitivity to both analogs. Thirty independent PutTnlOOO insertion mutants were tested for sensitivity to DHP and AZT. On the basis of their phenotypes, the put::Tn1000 mutants fell into two groups: (i) AZTSS DHPSS mutants and (ii) AZTr DHPr mutants (Table 3). The AZTSS DHPSS mutants have the phenotype expected for putA::Tn1000 putP+ plasmids: these mutants are supersensitive to AZT because they overexpress the putP gene and thus transport large amounts of the toxic analog. The putA::Tn1000 insertions cluster between 8.8 and 12.4 kb on the pKC7 physical map (Fig. 2). The AZTr DHPr mutants have the phenotype expected for putA putP::Tnl 000 plasmids: disruption of the putP gene prevents AZT transport, and any residual DHP transported by other permeases can be degraded by the putA gene product. The putP::TnlOOO insertions cluster between 13.0 and 14.2 kb on the pKC7 physical map (Fig. 2). These results suggest that the Klebsiella put operon is at least 5.2 kb in size and that the minimal sizes of the putP and

putA genes

are 1.2 and 3.6 kb, respectively. Furthermore, phenotypes of the putA insertion mutants indicate that, as in S. typhimurium and E. coli, the putA gene product regulates the expression of the Klebsiella putP gene. Expression of plasmid-encoded proteins. To identify the put gene products, we analyzed plasmids carrying the put genes in maxicells and in vitro transcription-translation assays (Fig. 3). A 140-kDa protein and a 38-kDa protein were expressed from the genes on pKC7 (putA+ putP+). These two proteins were not expressed from pKC14 (AputPA), a derivative of pKC7 with a deletion of most of the Klebsiella

the

DNA. A pKC7 derivative with a putA302::TnlOOO insertion mutation expressed the 38-kDa protein, but the 140-kDa protein was replaced by a truncated 25-kDa protein (data not shown). The 38-kDa protein was absent from plasmids with a putPl03::TnlOOO insertion mutation. These results indicated that, as in S. typhimurium and E. coli, the Klebsiella put operon encodes two proteins: the putA gene encodes a 140-kDa protein, and the putP gene encodes a 38-kDa

protein.

DNA sequence of the put regulatory region. Since the putA and putP genes in S. typhimurium and E. coli are transcribed divergently from a central regulatory region (19, 30), we expected that the organization of the Klebsiella put control region would be similar. The truncated peptides observed in maxicell analysis indicated that the direction of transcription of the Klebsiella putA gene is the same as that in E. coli and S. typhimurium. Therefore, we further characterized the region between the putP and putA structural genes. To identify potential regulatory sites, we determined the DNA sequence of the region between the putA and putP structural genes. Approximately 900 bp, including the regulatory region and the beginning of the putP and putA genes, was sequenced and compared with sequences from S. typhimurium and E. coli. As expected, the DNA sequences of the put control regions from Klebsiella strains, S. typhimurium, and E. coli were highly conserved (Fig. 4). Two methionine codons with appropriately spaced Shine-Dalgarno sequences were located 420 bp away from each other and divergently

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6.30

B Hindlil 1 Dral 12. Hindlil 12. EcoRI 1 2.2 Sail 11 .8! III

3.80

BamHl 1C

BamHI/Sall/EcoRl 5.30 Sail 7.85 EcoRI 7.10 FIG. 1. Restriction maps of plasmids pKM10 and pKC7. The numbers indicate map coordinates in kilobases. Plasmid pKC7 was obtained from a partial DraI digest of pKM10 which deleted most of the Mu A and B genes.

proceeded into open reading frames through the ends of the region sequenced. Transcriptional start sites of the putP and put4 genes. To identify the promoters used under nitrogen-limiting and

nitrogen-excess conditions, we determined the 5' ends of the putA and putP transcripts by primer extension analysis (Fig. 5). The putA gene was expressed from a single site located an appropriate distance downstream from a typical rpoD

TABLE 2. Expression of the put operona Strain

NH3 + succinate

KC1043 TR2619

TR2619(pKC7)

0.7 0.3 11.8

Proline oxidase activity with: NH3 + glucose NH3 + succinate + proline + proline

12.0 0.3 41.3

3.0

NDb ND

NH3 +

Glucose + proline

succinate

7.3 ND ND

0.011

Regulation of proline utilization in enteric bacteria: cloning and characterization of the Klebsiella put control region.

Enteric bacteria can grow on proline as the sole nitrogen and carbon source. Expression of the proline utilization (put) operon in Klebsiella strains ...
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