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Applied and Environmental Microbiology, November 2006, p. 7368-7372, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00294-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

In Vivo Insertional Mutagenesis in Corynebacterium pseudotuberculosis: an Efficient Means To Identify DNA Sequences Encoding Exported Proteins{triangledown}

Fernanda A. Dorella,1 Estela M. Estevam,1 Luis G. C. Pacheco,1 Cláudia T. Guimarães,2 Ubiraci G. P. Lana,2 Eliane A. Gomes,2 Michele M. Barsante,3 Sérgio C. Oliveira,3 Roberto Meyer,4 Anderson Miyoshi,1,{dagger} and Vasco Azevedo1*,{dagger}

Laboratório de Genética Celular e Molecular,1 Laboratório de Imunologia de Doenças Infecciosas, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte,3 Núcleo de Biologia Aplicada, Empresa Brasileira de Pesquisa Agropecuária, Sete Lagoas, Minas Gerais,2 Instituto de Ciências da Saúde, Vale do Canela, Salvador, Bahia, Brazil4

Received 2 February 2006/ Accepted 10 August 2006


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ABSTRACT
 
The reporter transposon-based system TnFuZ was used to identify exported proteins of the animal pathogen Corynebacterium pseudotuberculosis. Thirty-four out of 1,500 mutants had detectable alkaline phosphatase (PhoZ) activity. This activity was from 21 C. pseudotuberculosis loci that code for fimbrial and transport subunits and for hypothetical and unknown-function proteins.


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INTRODUCTION
 
Corynebacterium pseudotuberculosis, a gram-positive, facultatively intracellular pathogen, is the main etiological agent of caseous lymphadenitis (CLA), a common disease in sheep and goat populations throughout the world. CLA causes economic damage due to reduced wool and meat production, increased culling rates, and condemnations of carcasses and skins in abattoirs (5). Despite its importance for animal health, C. pseudotuberculosis is still poorly characterized, especially regarding genomic information. However, the genomes of several related species, such as Corynebacterium diphtheriae (2), Corynebacterium glutamicum (10), and Corynebacterium efficiens (16), have already been completely sequenced; this information will be helpful for better understanding of the biology of this microorganism.

We used a recently developed reporter transposon-based system, TnFuZ (8), to identify genes encoding exported proteins in C. pseudotuberculosis. This system combines a derivative version of the Tn4001 transposable element with the DNA fragment encoding the mature Enterococcus faecalis alkaline phosphatase gene (phoZ), whose product is active only when it is located outside the bacterial cytosol (8). Thirty-four out of 1,500 mutants had detectable PhoZ activity. We identified 21 loci coding for fimbrial and transport subunits, and also for hypothetical and unknown-function proteins, in C. pseudotuberculosis. These genes are potential targets for the development of new attenuated vaccine strains.


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In vivo insertional mutagenesis in C. pseudotuberculosis strain T1.
 
The C. pseudotuberculosis wild-type strain T1 was isolated from a caseous granuloma found in a CLA-affected goat in Bahia state (Brazil), identified by the API CORYNE battery (Biomerieux, France). Electrocompetent C. pseudotuberculosis cells were prepared (4) and transformed with 1 µg of the nonreplicative TnFuZ-containing plasmid (pCMG8). Insertional mutants were isolated by plating on selective brain heart infusion agar plates (Oxoid Ltd., England) containing 25 µg/ml of kanamycin, supplemented with 40 µg/ml of 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Sigma-Aldrich Co.), a substrate that allows recovery of C. pseudotuberculosis insertional mutant colonies with positive alkaline phosphatase activity (PhoZ+). We obtained 1,500 kanamycin-resistant C. pseudotuberculosis mutants, of which 34 (2.26%) exhibited the PhoZ+ phenotype.


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Molecular characterization.
 
After insertional mutagenesis, chromosomal DNA from the 34 selected PhoZ+ mutants was extracted by the 10% lysozyme and phenol-chloroform methods (18) and then directly sequenced using the Big Dye Terminator V3.1 cycle sequencing kit in an ABI 3100 automated DNA sequencer system (Applied Biosystems). The sequencing primer was EnPhoR1 (5'-TGC CTT CGC TTC AGC AAC CTC TGT TTG-3') (8), and the following PCR protocol was used: 4 min at 95°C and 100 cycles of 30 s at 95°C, 20 s at 50°C, and 4 min at 60°C. Sequences (approximately 200 bp) of interrupted C. pseudotuberculosis T1 genes from all 34 mutants were determined.

Nucleotide sequence similarity searches were performed with the BLAST software (http://www.ncbi.nlm.nih.gov/BLAST) service at the National Center for Biotechnology Information (NCBI). The nucleotide sequences were analyzed by searching DNA and protein databases for similarity with sequences of C. diphtheriae, C. efficiens, and C. glutamicum deposited in GenBank. Predicted amino-acid sequences were obtained by using the "Six Frame Translation Tool" service of the BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/seq-util/seq-util.html). Further analyses for the identification of exporting motifs were performed with the following bioinformatics programs: Pfam (http://www.sanger.ac.uk/Software/Pfam/search.shtml), SignalP (http://www.cbs.dtu.dk/services/SignalP-2.0/#submission), and PSORT (http://psort.nibb.ac.jp/form.html). Analyses of regions flanking the transposon insertion sites indicated similarity to 21 different loci, most of them encoding putative membrane proteins, such as fimbrial subunits and transport systems (Table 1). C. pseudotuberculosis DNA sequences encoding hypothetical and unknown-function proteins were also identified (Table 1).


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  		TABLE 1. Corynebacterium pseudotuberculosisDNA-PhoZ fusions

In on our analysis, 14 C. pseudotuberculosis mutants presented insertions in genes encoding cell envelope-associated proteins, 11 of which had insertions in different positions of the same locus of a putative fimbrial subunit found in C. diphtheriae NCTC13129 (2). This putative protein is a surface protein similar to the Actinomyces viscosus type 1 fimbrial major subunit precursor FimP. It is involved in bacterial binding to teeth through immobilized salivary statherin and acidic proline-rich protein; consequently, it participates in early plaque development and human mouth colonization (11). Fimbria and pilus proteins are particularly interesting, since fimbria-mediated adhesion is one of the best-studied strategies for host surface colonization by pathogenic microorganisms (6, 7, 13). Normally, these proteins play an important role in early steps of infection, since they are involved with pathogen-host adhesion. Bacterial adherence to host cells or surfaces is often an essential first stage in disease, because it places pathogens at appropriate target tissues. Adhesion to host cells may lead to internalization, either by phagocytosis or by bacterium-induced endocytosis (6, 7).

The DNA flanking the insertion from mutant CZ171053 encodes a putative-iron transport system binding (secreted) protein similar to that from C. diphtheriae NCTC13129 (2). This protein is similar to Escherichia coli FecB, which belongs to the bacterial solute-binding protein family and is involved in the transport of iron from ferric citrate (20). Proteins related to iron transport are utilized by many bacterial pathogens to perceive iron-limiting conditions of the host and as an environmental signal to induce expression of virulence factors (12, 17, 19). Moreover, iron modulates the adhesion of C. diphtheriae to cells of the human respiratory tract (15). In 2002, Billington et al. identified four genes in C. pseudotuberculosis involved in iron acquisition in mammalian hosts and concluded that this mechanism enhanced the capacity of this bacterium to develop a persistent infection in goats (1).

Two unknown genes were identified in the mutants CZ171068 and CZ171069. Although we obtained good-quality DNA sequences, ranging from 150 to 200 nucleotides, analyses of the DNA and amino acid sequences did not reveal any similarity with known sequences in databases. Searches for conserved protein motifs revealed probable cleavage sites in both of the deduced amino acid sequences (between amino acids 29 and 30 for CZ171068 and between amino acids 31 and 32 for CZ171069) of these proteins, indicating a hypothetical signal peptide; these will be investigated further.

The gene identified in mutant CZ171046 is similar to hypothetical protein NCgl2271 from C. glutamicum ATCC 13032 (10), which possesses a conserved cutinase domain (Pfam 01083; cutinase). Cutinase is a serine esterase, normally secreted by plant-pathogenic fungi, and it plays an important role in pathogenesis. It hydrolyzes plant cutin, thus facilitating fungus penetration (21, 22). Two cutinase-like proteins have also been found in the genome of Mycobacterium tuberculosis (3). Three mutants of this species (CZ171047, CZ171050, and CZ171062) have insertions in the same locus, a gene similar to that of a putative membrane protein of C. diphtheriae NCTC13129 (2); they have a conserved PspC (Pfam 04024; COG 1983). Proteins harboring this motif are associated with the cell envelope, functioning as a stress-responsive element (14).


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Phenotypic characterization by the alkaline phosphatase activity assay.
 
During the isolation and screening of C. pseudotuberculosis T1 insertional mutant colonies for the PhoZ+ phenotype, we observed two types of colonies: (i) "early-blue" colonies (25 out of 34 mutants), detected after 2 days of incubation, and (ii) "late-blue" colonies (9 out of 34 mutants), exhibiting a PhoZ+ phenotype after 3 to 4 days. The phenotypes were confirmed by streaking the colonies onto fresh plates.

We used the alkaline phosphatase assay (9) to monitor the activity level of alkaline phosphatase in filtered (pore size, 0.22 µm; Minisart; Sartorius Ltd., Epsom, United Kingdom) and unfiltered supernatants prepared from mid-log- to stationary-growth-phase bacterial cultures (optical density at 600 nm, 1.0 to 1.5). This approach was used to eliminate a possible background that could be generated by contamination with the remaining portions of the cells; it enabled differentiation between fusion proteins that were actually exported and those that normally remain in the cytoplasm. The alkaline phosphatase activities of C. pseudotuberculosis TnFuZ mutants were grouped into three categories, according to the proteins encoded by transposon-interrupted genes. (i) The first category consists of mutants harboring insertions in gene sequences that encode products homologous to exported proteins (Fig. 1a). As expected, this group had the highest levels of phosphatase alkaline activity, even after filtration. Although not all were secreted fusion proteins, we believe that the fimbrial subunit, for example, has weak interactions with the cell membrane and that this membrane is easily breached, making the fimbrial subunit detectable in the supernatant. (ii) The second category consists of mutants harboring insertions in gene sequences that encode products homologous to membrane proteins (Fig. 1b). This group has about 10-fold less alkaline phosphatase activity than the first group. When the supernatant is filtered, there is a significant reduction in alkaline phosphatase activity. Again, we believe that some fusion proteins bind weakly to the cell membrane and would be detectable in the supernatant, even after filtration. (iii) The third category consists of mutants harboring insertions in gene sequences that encode products homologous to cytoplasmic proteins (Fig. 1c). These fusion strains have only cell-associated alkaline-phosphatase activity. Only one protein was detectable, at moderate levels: a putative uroporphyrin III C-methyltransferase CysG, similar to that of C. efficiens. Since this putative protein is involved in coenzyme metabolism (as with COG 0007.2), being a cytoplasmic protein, it seems that the colony method of screening is not optimal for the identification of fusion proteins that do not remain associated with the cell surface, since it does not eliminate background signals.


Figure 1
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  		FIG.1. Quantitative alkaline phosphatase activity of Corynebacterium pseudotuberculosis TnFuZ mutants. Alkaline phosphatase activity was measured in filtered and unfiltered culture supernatants, as described in Material and Methods. Shown are results for mutants harboring insertions in gene sequences that encode products homologous to exported proteins (a), membrane proteins (b), or cytoplasmic proteins (c). Values are means of three independent experiments for which the supernatants were collected from the exponential-growth phase. T1, negative-control strain.


Figure 1
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  		FIG. 1— Continued.

This was the first time that the TnFuZ transposition system was used to identify genes coding for exported proteins of C. pseudotuberculosis. We identified a great diversity of proteins, including a fimbrial subunit, a protein related to iron uptake, adhesins, and proteins involved in transport, as well as hypothetical proteins and two unknown proteins. These data now constitute the largest collection of exported proteins identified in corynebacteria through genetic screening. Many of the genes that were identified could play an important role in the biology of C. pseudotuberculosis, and they are promising targets for the development of attenuated vaccine strains. Further experiments are now in progress in our laboratory in order to determine whether or not these exported proteins are involved in the virulence of this pathogen. We have also been conducting immunization assays to determine if these mutant strains can confer protective immunity against this bacterium.


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Nucleotide sequence accession numbers.
 
The ~200-bp nucleotide sequences of interrupted C. pseudotuberculosis T1 genes from the 34 selected PhoZ+ mutants have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html) under accession numbers CZ171041 to CZ171074.


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ACKNOWLEDGMENTS
 
We are grateful to Michel G. Caparon (Washington University Medical Center) for providing the TnFuZ-containing plasmid pCMG8 and to Philippe Langella, Yves Le Loir, Pascale Serror, and John Glen Songer for critical reading of various drafts of this paper.

This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil; PADCT/CNPq: 620004/2004-5), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), FINEP (Financiadora de Estudos e Projetos 01.04.760.00), and FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratório de Genética Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, CP 486, CEP 30161-970, Belo Horizonte, Minas Gerais, Brazil. Phone and fax: 55 31 34 99 26 10. E-mail: vasco{at}icb.ufmg.br. Back

{triangledown} Published ahead of print on 1 September 2006. Back

{dagger} V.A. and A.M. share credit for senior authorship of this work. Back


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Applied and Environmental Microbiology, November 2006, p. 7368-7372, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.00294-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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