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Cloning and characterization of the gene encoding the PepF endopeptidase from the aquatic bacterium Caulobacter crescentus

Clonagem e caracterização do gene que codifica a endopeptidase PepF da bactéria aquática Caulobacter crescentus

Abstracts

The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1) encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3), which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30% of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function.

endopeptidase; M3 family; gene regulation; bacteria


As metalopeptidases têm papel importante em bactérias, estando envolvidas em vários processos que dependem de reciclagem de proteínas, como nutrição, degradação de peptídios-sinal, localização de proteínas e virulência. Nós clonamos e caracterizamos o gene da metaloendopeptidase PepF da bactéria aquática Caulobacter crescentus. O gene a montante de pepF (orf1) codifica uma proteína hipotética conservada encontrada em Mycobacterium e Streptomyces. pepF é co-transcrito com o gene a juzante (orf3), que codifica uma proteína pertencente à família ABC1 de proteínas quinases, sugerindo que estas duas proteínas podem compartilhar uma função comum na célula. A proteína PepF de C. crescentus possui o motivo HEXGH presente nos domínios de ligação de zinco de homólogos de PepF. A interrupção do gene pepF por inserção de sequência de DNA de um vetor não gerou nenhum defeito de crescimento, mas a linhagem mutante possui apenas 30% da atividade especifica de endopeptidases presentes na linhagem selvagem. Deleções e mutações pontuais na região regulatória mostraram que há duas possíveis regiões promotoras, e a expressão do operon é independente do regulador de transcrição CtrA. Os resultados indicam que PepF não é essencial para o crescimento desta bactéria utilizando peptídios como fonte de carbono, sugerindo que outras peptidases possam estar realizando esta função.

endopeptidase; família M3; regulação gênica; bactéria


CLONING AND CHARACTERIZATION OF THE GENE ENCODING THE PEPF ENDOPEPTIDASE FROM THE AQUATIC BACTERIUM CAULOBACTER CRESCENTUS

Vânia S. Braz; Elza A.S. Lang; Marilis V. Marques* * Corresponding author. Mailing address: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo. Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brasil. Phone: (+5511) 3091-7299. FAX: (+5511) 3091-7354. E-mail: mvmarque@usp.br

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil

Submitted: December 05, 2001; Returned to authors for corrections: January 09, 2002; Approved: February 25, 2002

ABSTRACT

The metallopeptidases have a very important role in bacteria, being involved in several processes that rely on protein turnover, such as nutrition, degradation of signal peptides, protein localization and virulence. We have cloned and characterized the gene of the metalloendopeptidase PepF from the aquatic bacterium Caulobacter crescentus. The gene upstream of pepF (orf1) encodes a conserved hypothetical protein found in Mycobacterium and Streptomyces. pepF is co-transcribed with the gene downstream (orf3), which encodes a protein that belongs to the ABC1 protein kinase family, suggesting that these two proteins may share a common function in the cell. The C. crescentus PepF protein possesses the conserved HEXGH motif present in zinc binding domains of PepF homologs. Disruption of the pepF gene by insertion of a vector sequence did not produced any growth defect, but the mutant strain possesses only 30% of the specific activity of endopeptidases present in the wild type strain. Deletions and point mutations in the regulatory region showed that there are two putative promoter regions, and the operon expression is independent of the transcription regulator CtrA. The results indicate that PepF is not essential for either growth or development of this bacterium using peptides as the sole carbon source, suggesting that other peptidases can be sharing this function.

Key words: endopeptidase, M3 family, gene regulation, bacteria

INTRODUCTION

Bacterial peptidases are involved in several important processes in bacterial cells, such as protein localization, hydrolysis of imported peptides, protein turnover and virulence. The metallopeptidases comprise a large group of proteins and are categorized in several families, according to their sequence similarity and function (22). The zinc-dependent metallopeptidases are found in bacteria, protozoa, fungi, plants and animals, and share a common amino acid motif that comprises the zinc-binding site (11). The PepF peptidases are included into family M3, and this class of peptidases is found in all organisms except for viruses and the archaebacteria.

PepF homologs have been found in both Gram negative and Gram positive bacteria, but the role of this protein in the cell was determined only in Lactococcus lactis, where two pepF genes were found, one encoded in a plasmid and one in the chromosome (16,17). In L. lactis, PepF is involved in the ability of the bacterium to utilize milk casein as a carbon source, where it is responsible for degradation of peptides from 7 to 17 amino acids in size (16). The loss of the plasmid copy of pepF leads to a 75% reduction in total endopeptidase activity, indicating that this oligopeptidase is responsible for most of this activity in the cell (16).

The aquatic bacterium Caulobacter crescentus presents an obligatory differentiation step in each cell cycle, generating by cell division a motile swarmer cell and a sessile stalked cell (3,9). Its unique developmental program has been extensively studied, several key regulatory genes have been identified, and proteolysis plays an important role in regulating C. crescentus development (10,25). Despite being a heterotroph, Caulobacter has a preference for growing in very dilute environments, which implies having an efficient way of taking organic matter, and therefore oligopeptidases must have an important role in this bacterium. The recently completed genome sequencing of C. crescentus (19) showed 48 genes encoding putative peptidases that belong to different families, but only one was predicted to be from the M3 family, a PepF homolog. In this report, we describe the genetic organization of the locus encoding the only pepF homolog found in the genome from C. crescentus and characterize its regulatory regions.

MATERIALS AND METHODS

Bacterial strains and media

Caulobacter NA1000 is the wild type, synchronizable strain (7) and the LS2195 strain has a temperature-sensitive allele of the ctrA gene (21). E. coli S17-1 contains the mobilization genes that allow DNA transfer by conjugation to C. crescentus (23). Caulobacter strains were grown at 30ºC in PYE complex media or M2 minimal media (6), as indicated.

Cloning and characterization of the pepF locus

The first half of the pepF gene was identified in a clone previously isolated in our laboratory (14). To obtain the complete region containing pepF gene, two partial libraries were constructed in vector pUCBM20 (Boehringer Mannheim) by digestion of the original cosmid clone, one library containing HindIII/PstI fragments and the other containing SacI/BamHI fragments. The first library was screened with radioactive probes corresponding the beginning of pepF. A positive HindIII/PstI clone of 2.4 kb was obtained and its DNA sequence was determined after subcloning into M13mp18 (Gibco BRL). The nucleotide sequence of this clone showed it contained the complete pepF coding region, encompassing 1.6 kb, and part of a third ORF (ORF3). A SacI/BamHI clone of 2.3 kb was isolated, by hybridization with a probe corresponding to the beginning of ORF3. This clone was completely sequenced, showing that ORF3 is 1.2 kb long and no other ORF could be identified immediately downstream of it, using the GeneMark algorithm (1) (Fig. 1).


RT-PCR

In order to determine whether the three genes are organized in an operon, RT-PCR reactions using 1 µg of total RNA and primers flanking each intergenic region were performed with the SuperScript One-Step RT-PCR kit (Gibco BRL). RT-PCR between orf1 and pepF was performed using the pair of primers RT-1/RT-2 (RT-1: 5'-gcgatggcctggagctatc-3', RT-2: 5'-actagcagataggcctccg-3'), and between pepF and orf3 was performed using the pair RT-3/RT-4 (RT-3: 5'-gccgctttatgagcaactgc-3', RT-4: 5'-ggttggcaccg taagacac-3'). As a negative control for each RT-PCR, the reverse transcriptase was ommited from the reactions.

Mapping of regulatory regions

In order to define more precisely the regulatory sequences involved in pep expression, different promoter constructions were cloned in front of a reporter lacZ gene (plasmid pRKlacZ290, (8)), and the levels of expression were determined by measuring b-galactosidase activity (15). Site-directed mutagenesis was performed using primer tac3 (5'-GCATAAGACCCTGTTA AGCCTAAG-3') or tac7 (5'-TCCGAAAGCCGGTGTTTA CCCGCGACCTG-3') as described (12). pVB04 was obtained by cloning the 0.37 kb HindIII/StuI fragment containing part of the orf1 gene into vector pRKlacZ290. pVB01 was obtained by PCR using primers tac5 (5'-TCGCGCTCGAGCACCGCTTGA-3') and V2 (5'-CAGCACGCCGGGATCCAGCACGCGCGC-3').

Construction and analysis of a pepF/orf3 mutant

To obtain a null mutant of the pepF gene, a 0.65 kb SacI/StuI fragment corresponding to the central region of the gene was cloned into plasmid pNPTS138. The recombinant plasmid was introduced by electroporation into E. coli S17-1 and the cells were plated in LB/Km plates. The plasmid was transferred to Caulobacter strain NA1000 by conjugation (6). To confirm the correct integration of pNPTS138 in the pepF locus, genomic DNA from the transformed clones was purified (5), digested with SacI and transferred to a nylon membrane. The correct integration of the plasmid was confirmed by hybridization with radioactive probes corresponding to either the pNPTS138 plasmid or to the 0.65 kb SacI/StuI fragment. The mutant strain was designated SP2008.

Determination of endopeptidase activity

Endopeptidase activity was assayed in C. crescentus strains NA1000 and SP2008 grown to midlog phase. Cells were separated from the culture medium by centrifugation for 15 minutes at 7500 x g, and ressuspended in buffer A (50 mM Tris-HCl pH 7.0/ 20 mM NaCl). Cells were broken by sonication (four pulses of 15 seconds in a Branson Sonifier 250), and cell debris was removed by centrifugation for 5 minutes at 12,000 x g. The enzymatic assay of either cell extracts or culture supernatants was carried out as described (20) in a final volume of 1 ml of buffer A, containing or not 0.5 mM DTT, and the quenched fluorogenic peptide substrate QF7 (orto-aminobenzoyl (Abz)-Gly-Gly-Phe-Leu-Arg-Arg-Val-(2,4,dinitrophenyl) ethylenediamine (EDDnp). Protein concentration was determined by the Bradford assay (BioRad).

RESULTS AND DISCUSSION

Sequencing of the pepF gene region showed that it is preceded by a small ORF (ORF1) and that downstream there is another ORF 1.2 kb long (ORF3) which is separated of pepF by only one base pair (Fig. 1). Our results were later confirmed by the complete genome sequencing, where the genes correspond to numbers CC3314 (orf1), CC3313 (pepF) and CC3312 (orf3) in the TIGR database (see http://www.tigr.org).

In order to determine whether the three genes are organized in an operon, RT-PCR reactions using primers flanking each intergenic region were performed. As shown in Figure 2, an amplification product is obtained between pepF and ORF3, only when the reverse transcriptase is present, while the negative control without this enzyme did not show any amplified band. This result shows that these two genes are transcribed in a single mRNA species. Confirming this result, no promoter activity was observed when the 1.2 kb XhoI/StuI restriction fragment between pepF and ORF3 was cloned in front of the reporter gene lacZ, indicating that there is no promoter in this region (not shown). RT-PCR between ORF1 and pepF (Fig. 2, lane 2) produced a very faint band, although specific, and this result could not be improved after several attempts. The intergenic space between orf1 and pepF is small (41 bp); no putative regulatory sequences could be identified in this region; and there is no intrinsic transcription terminator between orf1 and pepF. These results altogether indicate that orf1 is co-transcribed with pepF and orf3.


The deduced amino acid sequences of the three ORFs were compared to the available databases. ORF1 is similar to hypothetical proteins from Mycobacterium tuberculosis (35% identity) and Streptomyces coelicolor (39% of similarity). PepF presents strongest similarity to PepF from Helicobacter pilori (33% identity) and from Lactococcus lactis (29% identity) (Fig. 3). A domain analysis of PepF showed that it presents a common domain to several other metallopeptidases (PRODOM domain PD005933), sharing with these proteins the zinc-binding region signature (HEXGH) (22), and it also contains a putative transmembrane domain from amino acids 71 to 91.


ORF3 was annotated as a conserved hypothetical protein in the genome analysis (19), but a further analysis showed that it belongs to the large ABC1 protein kinase family, which includes representatives both in eukaryotes and prokaryotes (13). ORF3 presents strongest similarity to a hypothetical protein from S. coelicolor (29% identity), to a hypothetical protein from C. elegans (28% identity), to hypothetical protein Rv3197 from M. tuberculosis (32% identity), to a putative ABC transporter from Methanobacterium thermoautotrophicum (29% identity) and to several eukaryotic ABC1 homologs. The ABC1 family includes several eukaryotic proteins that are essential for the efficient functioning of the mitochondrial bc1 complex, as well as the E. coli ubiB gene, that is required for ubiquinone biosynthesis (2). The pepF gene disruption in strain SP2008 (see below) could lead to a polar effect in the orf3 gene, since no promoter activity was detected between these two genes. Caulobacter is an obligatory aerobic prokaryote, therefore any mutations that impair respiration should be deleterious to the cell. Strain SP2008 shows no growth defect that could indicate a loss of respiratory function, which suggests that the role of ORF3 is probably not related to respiratory chain function.

A pepF null mutant was constructed by disrupting the gene with a plasmid encoding a kanamycin resistance marker, the insertion of the plasmid in the pepF locus was confirmed by Southern blotting (not shown). The pepF::pNPTS138 strain (SP2008) showed no growth defects, with a doubling time of 3 h, equivalent to the wild type strain NA1000, and normal swimming behaviour was observed by light microscopy. To assess whether a pepF/orf3 mutation would have an effect on survival under stress conditions, a nutritional stress was induced by allowing the cells to enter stationary phase, and cell viability was determined. SP2008 showed no reduction in viability during the stationary phase when compared to strain NA1000, indicating that PepF and ORF3 are not essential for cell viability during prolonged growth. Strain SP2008 is also able to grow in peptone/yeast extract medium, indicating that PepF activity is not essential for nutrition under these conditions.

Since the pepF gene encodes a peptide highly similar to metalloendopeptidases of the M3 family, endopeptidase activity of strain SP2008 was assayed towards a peptide that is a substrate for the thimet mammalian oligopeptidases (20). In order to have an indication whether PepF localization is cytoplasmatic or extracellular, endopeptidase activity was determined in the culture supernatants as well as in total cell extracts. Some mammalian metalloendopeptidases that belong to the M3 family are the so-called thimet peptidases, whose activity is dependent on thiol (22). Activity was assayed using a quenched peptide substrate that becomes fluorescent when cleaved by an endopeptidase, in the presence and absence of 0.5 mM DTT. As observed in Table 1, the supernatants of both NA1000 and SP2008 cultures showed a similar specific activity, indicating that a similar set of enzymes are secreted in both cases. The absence of PepF activity in the cell culture medium does not support the hypothesis of the peptidase being secreted, and so it is probably localized in the cytoplasm or associated with the cell membrane. However, endopeptidase activity measured in cell extracts using this substrate, showed a 30% reduction on specific activity of strain SP2008 when compared to strain NA1000, suggesting that disruption of pepF caused a loss of a significant cellular endopeptidase activity in the mutant. This result also indicates that there are other peptidases in the cell that are able to cleave this substrate, which agrees with the presence of so many genes encoding peptidases in the genome (19). Forty-eight gene products were annotated as putative peptidases, out of which nine are from the M20/25/40 families, seven from the M23/27 families, four from the M16 family and one representative of each of the M1, M3, M13, M22 and M24 families.

An increase in activity was observed when the assays were carried out in the absence of DTT in all cases, indicating that even if there is a thiol-dependent endopeptidase in Caulobacter, its activation is not very pronounced, and some of the enzymes are in fact inhibited by thiol. Our results show that C. crescentus PepF hydrolyzes the peptide of 7 residues that is a substrate for the mammalian thimet oligopeptidase (4). The L. lactis PepF is also capable of hydrolyzing peptides very similar to this, containing 7 to 17 amino acids, with a cleavage site preference after a Phe residue (16). The substrate specificity of C. crescentus PepF has not been determined, but since it is similar to the L. lactis enzyme, some of its catalytic properties should be conserved.

The promoter region of the pep operon shows several notable features (Fig. 1). Upstream of the divergent tacA transcription start site there is a binding site for the global transcription regulator CtrA (GAAA-N7-TTTAC; (21)) centered 25 bp upstream of the tacA start site (14). The region upstream of orf1 contains one inverted repeat that could have a regulatory role, centered at position -18 relative to orf1 most upstream ATG. Two alternative start codons for orf1 are located 15 bp downstream, and at least the first of these has a good ribosome binding site consensus sequence.

The expression of pep in two C. crescentus strains was determined through b-galactosidase activity assays in cells containing the pMV102 construction (Fig. 4A). This plasmid was introduced into strain NA1000 (wild type) and into strain LS2195, in which the CtrA protein is inactive at the temperature of 37ºC. In order to investigate whether pepF transcription was somehow affected by CtrA, its expression was determined by b-galactosidase activity assay both at 30ºC and at 37ºC. Figure 4A shows that the levels of b-galactosidase activity were even a little higher at 37ºC, indicating that pepF expression is not affected by the absence of CtrA.



The RT-PCR results have indicated that two distinct promoters could be used to transcribe the pepF gene, one before orf1 (P1) and another between orf1 and pepF (P2). A two-base alteration in the inverted repeat localized at position ¾18 (from orf1 first ATG) did not alter the levels of expression, indicating that this sequence does not have a regulatory role. In order to analyze each promoter separately, deletions were made both upstream and downstream the orf1 gene (Fig. 4B). A deletion of the region upstream the HindIII site (pVB04) caused a little pronounced reduction in the b-galactosidase levels, suggesting that the promoter P2 between orf1 and pepF is responsible for most the transcription activity. This can be also confirmed by deletion of downstream sequences, indicating that the P1 promoter is not very strong; the construction that removes the region downstream the beginning of orf1 confers less than half the activity of b-galactosidase (compare pVB01 and pMV102). A mutation that inactivates the CtrA binding site has no effect in P1 activity (pVB07), but the two-base alteration in the inverted repeat causes a reduction in expression (pMA03). The results with pVB04 indicate that the region comprised between the HindIII site and the StuI site contains promoter activity, which is probably responsible for transcription of the pepF/orf3 genes, explaining the increased amount of mRNA corresponding to pepF and orf3 observed in the RT-PCR.

The structure of the pep operon in C. crescentus is very interesting, since the pepF gene is co-transcribed with a gene encoding a putative protein kinase, which could indicate a coordinate role for the two peptides in the cell. The absence of cell cycle regulation indicates that this endopeptidase is probably involved in basal metabolic functions, as described for L. lactis (16,17). However, we could see no gene induction by the presence of peptides in the culture medium, and the pepF mutant strain is able to grow using peptides as the sole carbon source (not shown), suggesting that there could be other peptidases in the cell which are responsible for nutrition.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. Suely L. Gomes for her help throughout this work and for the critical reading of the manuscript and Dr. A.C.M. Camargo and people from his lab for their help with the endopeptidase assays. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). During the course of this work, V.S.B. was supported by fellowships from FAPESP and from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

RESUMO

Clonagem e caracterização do gene que codifica a endopeptidase PepF da bactéria aquática Caulobacter crescentus

As metalopeptidases têm papel importante em bactérias, estando envolvidas em vários processos que dependem de reciclagem de proteínas, como nutrição, degradação de peptídios-sinal, localização de proteínas e virulência. Nós clonamos e caracterizamos o gene da metaloendopeptidase PepF da bactéria aquática Caulobacter crescentus. O gene a montante de pepF (orf1) codifica uma proteína hipotética conservada encontrada em Mycobacterium e Streptomyces. pepF é co-transcrito com o gene a juzante (orf3), que codifica uma proteína pertencente à família ABC1 de proteínas quinases, sugerindo que estas duas proteínas podem compartilhar uma função comum na célula. A proteína PepF de C. crescentus possui o motivo HEXGH presente nos domínios de ligação de zinco de homólogos de PepF. A interrupção do gene pepF por inserção de sequência de DNA de um vetor não gerou nenhum defeito de crescimento, mas a linhagem mutante possui apenas 30% da atividade especifica de endopeptidases presentes na linhagem selvagem. Deleções e mutações pontuais na região regulatória mostraram que há duas possíveis regiões promotoras, e a expressão do operon é independente do regulador de transcrição CtrA. Os resultados indicam que PepF não é essencial para o crescimento desta bactéria utilizando peptídios como fonte de carbono, sugerindo que outras peptidases possam estar realizando esta função.

Palavras-chave: endopeptidase, família M3, regulação gênica, bactéria

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  • *
    Corresponding author. Mailing address: Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo. Av. Prof. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brasil. Phone: (+5511) 3091-7299. FAX: (+5511) 3091-7354. E-mail:
  • Publication Dates

    • Publication in this collection
      16 Sept 2002
    • Date of issue
      Jan 2002

    History

    • Accepted
      25 Feb 2002
    • Received
      05 Dec 2001
    • Reviewed
      09 Jan 2002
    Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
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