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Preliminary analysis of glutathione S-transferase homolog fromLactobacillus casei Zhang

Preliminary analysis of glutathione S-transferase homolog fromLactobacillus casei Zhang Annals of Microbiology, 59 (4) 727-731 (2009) Preliminary analysis of glutathione S-transferase homolog from Lactobacillus casei Zhang 1§ 2,3§ 1 1 2,3 4 Wen Yi ZHANG , Dong Liang YU , Zhi Hong SUN , Caicike AIRIDENG , Song Nian HU , He MENG *, He Ping ZHANG * Key Laboratory of Dairy Biotechnology and Engineering Ministry of Education, College of Food Science and Engineering, Inner Mongolia Agricultural University, Huhhot 010018; Key Laboratory of Genome Science and Information, Beijing In- stitute of Genomics, Chinese Academy of Sciences, Beijing 100029; James D. Watson Institute of Genome Sciences, Zhe- jiang University, Hangzhou 310029; School of Agriculture and biology, Shanghai Jiao Tong University, Shanghai 200240, China Received 12 June 2009 / Accepted 28 September 2009 Abstract - Glutathione S-transferases (GSTs) constitute a large protein family. Some of them are proved to be associated with biphenyl metabolism. In this study, we described a homolog of GST gene in Lactobacillus casei Zhang that revealed by genome analysis. The GST gene itself as well as the adjacent gene context was highly conserved among different L. casei isolates. Phylogenetic investigation suggested that the GST gene was acquired via horizontal gene transfer event, possibly from gamma subdivision. Additional survival experiment indicated that it may not benefit the L. casei host for growing in the presence of biphenyl. It was for the first time discovered that GST was encoded by Lactobacillaceae species. Key words: Lactobacillus casei Zhang; glutathione S-transferase; adjacent genes; horizontal gene transfer. INTRODUCTION of China (Zhang et al., 2008a). Attempting to track its potential biological role, distribution, phylogenetic, and preliminary func- Bacterial glutathione S-transferases (GSTs, EC 2.5.1.18) con- tion analysis were performed. stitute a large protein family with functional versatility as which in eukaryotic organisms, such as association with aromatic compounds metabolism and response to oxidative stress (Lloyd- MATERIALS AND METHODS Jones and Lau, 1997; Fortin et al., 2006). Hitherto they have been broadly identified and characterized in bacterial strains, and Bacterial strains and culture conditions. Lactobacillus casei grouped into four main classes (Allocati et al., 2008). Several Zhang and other fifty-eight Lactobacillus casei isolates were studies have been carried out to assess the potentials of the GSTs obtained from Key Laboratory of Dairy Biotechnology and using both proteins and microorganisms engineering (Kurtovic Engineering Ministry of Education, Inner Mongolia Agricultural et al., 2008; Yan et al., 2008). Nevertheless, experimental and University, China. They were all identified by conventional and biotechnological investigations appeared to be undertaken within 16S rRNA methods (data unpublished). Lactobacillus casei a set of bacterial species. Firmicutes strains encoded GSTs have strains were propagated at 37 °C in Trypticase peptone yeast not been described commonly as which in Proteobacteria or broth (TPY; DIFCO, PQ, Canada). Cyanobacteria (Vuilleumier, 1997). GSTs encoded by strains of Streptococcaceae and Carnobacteriaceae have been identified, PCR amplification and sequencing. Total genomic DNA was yet did not any from Lactobacillaceae (Vuilleumier and Pagni, extracted by using Bacterial DNA isolation kit (Tiangen, Beijing, 2002). China) according to manufacturer’s instructions. Based on the In the present study, we described a homolog of GST gene finished genome sequences of L. casei Zhang, a primer pair identified in the genome of Lactobacillus casei Zhang, a new pro- (GST_F, GST_R) that designed within the GST gene was used biotic strain isolated from home-made koumiss in Inner Monglia to detect its existence. Two primer pairs (GST_L3F, GST_L3R; GST_L4F, GST_L4R) that placed in the upstream genes, encoding a saccharopine dehydrogenase related protein and a transcrip- tion regulator, were used to investigate its adjacent gene context These authors contributed equally to this work. among different L. casei isolates. Primers are listed in Table 1. * Corresponding Authors. H.P. Zhang - Phone: 86-471-4319940; PCR reaction was carried out in a 20 μl reaction mixture con- Fax: 86-471-4300122; E-mail: hepingdd@vip.sina.com; taining 0.2 μl Taq polymerase (5 U/μl, Takara Tokyo, Japan), 2.5 H. Meng - Phone: 86-21-34206145; Fax: 86-21-34204538; 2+ μl 10X PCR Buffer (Mg free), 2 μl dNTPs (2.5 mM each), 2 μl E-mail: menghe@sjtu.edu.cn 728 W.Y. ZHANG et al. TABLE 1 - PCR primers used package with neighbor-joining method (Tamura et al., 2007). Reliability of the nodes was tested with 1000 repetitions. Primer Sequence (5′-3′) Microbial counts in the presence of biphenyl. Defined GST_F TACGATCAAATCAACGCCA medium containing 10 g proteose peptone, 5 g tryptone, 3 g GST_R CGCAGAACTTCCTGCACAC yeast extract, 1 g Tween-80, 5 ml solution B (10 g MgSO ·7H O, 4 2 0.5 g FeSO ·7H O, 0.5 g NaCl, 0.337 g MnSO , 250 ml ddH O), GST_L3F ACAATTAGGAAAAGAACACGG 4 2 4 2 0.2 g L-cyctein HCl, and 1000 ml Bacto-Liver extract was used GST_L3R GCTCGAAAATGATACGACACT for strain cultivation (Kozaki et al., 1992). Overnight cultures of L. casei Zhang were inoculated in the defined medium and the GST_L4F GTTAGTGACTGCCATCGTGAGA defined medium supplemented with 0.5 mM biphenyl respec- GST_L4R AAGGCGAAATAAAGTTTTTGTG tively (Bartels et al., 1999). After incubation at 37 °C for 0, 12, 36, and 48 h, survivors were enumerated on TPY agar plates in triplicate (Zhang et al., 2008b). MgCl (25 mM), 1 μl forward primer (1 pM), 1 μl reverse primer (1 pM), 1 μl genomic DNA (100 ng/μl), and 10.3 μl ddH O. Nucleotide sequence accession numbers. The sequence of Amplifications were performed on a thermal cycler (Bio-Rad, L. casei Zhang GST gene is contained in its genome sequences, California, USA) as following program: 94 °C for 5 min, 94 °C for accession number CP001084. Ten partial sequences of GST gene 30 s, 56.5 °C for 30 s, 72 °C for 40 s, 45 cycles, then 72 °C for and corresponding adjacent genes of L. casei strains have been 10 min, 4 °C for 30 min. deposited in GenBank under the accession numbers of FJ750469- The PCR products were separated by agarose gel electro- FJ750478. phoresis, extracted using a Huashun Gel Extraction Kit (Huashun, Shanghai, China) and directly sequenced. Fragment sequences were assembled to contigs with EditSeq program of DNAstar RESULTS AND DISCUSSION software (DNASTAR, Madison, WI, US). PCR amplification and sequencing Analysis of the sequences. Domain characterization was Analysis of L. casei Zhang genome has identified a homolog carried out by using Interpro database (Mulder et al., 2005). of GST gene with a full-length of 630 bp (LCAZH_1208). GST Homolog searching was performed against nr database provided N-terminal (PF02798) and C-terminal (PF00043) domains were by NCBI website using BLASTP (Altschul et al., 1997), and the found in its deduced protein sequence. To further explore the most similar sequences were selected for phylogenetic analysis conservation properties of the GST sequences distributed in with the cutoff E-value 1e-3 and the identity 25%. Jalview 2.3 genus L. casei, we did the screening tests among different iso- was used to remove the redundancy of the sequences (Clamp lates by using standard PCR. Upstream regions of the GST gene et al., 2004). Phylogenetic tree was reconstructed using MEGA were determined and sequenced as well. FIG. 1 - Electrophoretic characterization of PCR amplification products of GSTs from fifty-nine different Lactobacillus casei isolates. M: DL2000 maker; lane 1-59: L. casei strain Zhang, 1301, 1302, 1303, 1305, 1307, 2303, 4305, 6301, 6304, 9303, 11301, 13303, 14302, 14303, 14306, 15301, 15302, 15304, 15305, 15306, 23302, 24304, 25302, 25303, 25304, 29301, 29303, 29305, 33304, 29307, 29306, 33303, 29308, 34303, 34304, 34306, 37302, 37303, 39305, 41303, 43301, 43302, 43306, AG8-2, AG8-3, AG8-5, AG9-1, AG9-2, AG9-4, AG9-5, XM2-1, XM7-1, ZL3-1, ZL3-2, AZ43-3-4-2, AY2-2, AZ43-3-4-1, and ZL3-3. Ann. Microbiol., 59 (4), 727-731 (2009) 729 FIG. 2 - Phylogenetic estimation of the relationship between different bacterial GSTs. Bootstrap values were reported for greater than 40% and were shown at each of the corresponding nodes. Fifty-nine isolates from five distinct regions of China were gene contexts with L. casei Zhang, a saccharopine dehydroge- employed during PCR trials. Amplifications within the DNA nase related protein and a transcription regulator were detected template of L. casei Zhang were used for control purpose. As upstream the GST. Likewise the GST detected in Escherichia coli presented in Fig. 1, except four strains (11301, 29306, 29308, K-12, it was neither part of the gene clusters of bph nor flanked and az43-3-4-2), all experimental strains represented the same by genes relevant to catabolic functions (Blattner et al., 1997). amplification pattern. Sequencing ten of the total PCR products revealed > 98% similarity with homologous regions in L. casei Phylogenetic analysis Zhang. This could indicate that the GST gene is relatively con- A gene-based tree was constructed so as to trace the origin of served among the members of genus L. casei, which suggests an GST gene identified in L. casei Zhang. Representative GST mem- important physiological role. bers from thirty-seven different bacterial species were used dur- Additional PCR reactions were performed to precisely exam- ing the phylogeny test (Fig. 2). However, owing to the low level ine gene organizations upstream the GST of previously selected of identity between different bacterial GSTs, only a few nodes are isolates. Similarly results were also obtained as analyzing the supported by high bootstrap values. The real evolution history of GST distribution. Sequences of these PCR products using prim- these GSTs is still uncertain, but the phylogenetic analysis partly ers GST_L3F and GST_L3R, or GST_L4F and GST_L4R showed > indicates the close relationship of L. casei Zhang encoded GST 96% similarity with L. casei Zhang. The ten isolates had identical gene with its counterparts from Firmicutes. 730 W.Y. ZHANG et al. FIG. 3 - Survival of Lactobacillus casei Zhang cells during the 48 h continuous cultivation: ◆ Biphenil-free, ■ Biphenil treatment. It can be observed from the main topology in the tree, most, REFERENCES if not all of the well-defined clusters are consisted of GST mem- bers from the same taxon. Among them, Escherichia coli as well Allocati N., Federici L., Masulli M., Di Ilio C. (2009). Glutathione as other five Enterobacteriales GSTs was shown as the largest transferases in bacteria. FEBS J., 276: 58-75. cluster. L. casei GST was grouped with Enterococcus faecalis Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., encoded GST, appeared most close to those from gamma subdi- Miller W., Lipman D.J. (1997). Gapped BLAST and PSI-BLAST: vision bacteria. Indeed, GSTs have been identified in strains from a new generation of protein database search programs. Lactobacillales, but no protein belonged to GST family has been Nucleic Acids Res., 25: 3389-3402. detected in Lactobacillaceae species. These findings indicated Bartels F., Backhaus S., Moore E.R., Timmis K.N., Hofer B. that GST encoded by L. casei might acquire from the horizontal (1999). Occurrence and expression of glutathione-S-trans- gene transfer event. ferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria. Microbiology, 145: Preliminary function assay 2821-2834. Since GST information of dairy-related bacteria has not been reported, a greedy search was performed with BLAST. Analysis Blattner F.R., Plunkett G., 3rd Bloch C.A., Perna N.T., Burland V., results indicated that GST encoded by L. casei Zhang was a novel Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew gene, no homologous gene can be found in any other sequenced G.F., et al. (1997). The complete genome sequence of Lactobacillus genomes. The putative GST protein shares 37% Escherichia coli K-12. Science, 277: 1453-1474. identity to BphK encoded by Burkholderia xenovorans LB400. Clamp M., Cuff J., Searle S.M., Barton G.J. (2004). The Jalview Most often, it was found located downstream of the gene clusters Java alignment editor. Bioinformatics, 20: 426-427. encoding degradation pathway of biphenyl (bph) (Ohtsubo et al., Fortin P.D., Horsman G.P., Yang H.M., Eltis L.D. (2006). A glutath- 2001). Bartels et al. (1999) has demonstrated that BphK was ione S-transferase catalyzes the dehalogenation of inhibitory able to fulfill the function of biphenyl (BP) utilization to their host metabolites of polychlorinated biphenyls. J. Bacteriol., 188: organisms, although it was not essential. 4424-4430. To test whether the GST gene can be an advantage for L. casei Zhang in the presence of biphenyl, viability of the cells Kozaki M., Uchimura T., Okada S. (1992). Laboratory Manual of was measured. After 48 h consecutive incubation, total viable Lactic Acid Bacteria, Asakura Book Store, Tokyo. cells of L. casei Zhang in the biphenyl-free medium increased, Kurtovic S., Shokeer A., Mannervik B. (2008). Diverging catalytic which ranged from 8.83 to 9.78 log CFU/ml (Fig. 3). On the capacities and selectivity profiles with haloalkane substrates contrary, cells propagated in the biphenyl treatment medium not of chimeric alpha class glutathione transferases. Protein Eng. only presented a slow growth rate but also seemed decreased Des. Sel., 21: 329-341. at some level during the latter 36 h. Therefore, we tentatively Lloyd-Jones G., Lau P.C. (1997). Glutathione S-transferase- propose that the cells of L. casei Zhang may not have the ability encoding gene as a potential probe for environmental bacte- to utilize biphenyl. However, this does not reflect the useless role rial isolates capable of degrading polycyclic aromatic hydro- GST played in its host L. casei strain. Further studies about GST carbons. Appl. Environ. Microbiol., 63: 3286-3290. should be performed for demonstrating its biological roles in L. casei. Mulder N.J., Apweiler R., Attwood T.K., Bairoch A., Bateman In conclusion, we identified and characterized a homolog of A., Binns D., Bradley P., Bork P., Bucher P., Cerutti L., et al. GST gene encoded by L. casei Zhang genome. Although it was (2005). InterPro, progress and status in. Nucleic Acids Res., done within a preliminary frame, analysis results from distribu- 33: D201-205. tion, phylogenetic and function tests make it possible for future Ohtsubo Y., Delawary M., Kimbara K., Takagi M., Ohta A., Nagata associated works. Y. (2001). BphS, a key transcriptional regulator of bph genes involved in polychlorinated biphenyl/biphenyl degradation Acknowledgements in Pseudomonas sp. KKS102. J. Biol. Chem., 276: 36146- This research was supported by National Natural Science Foundation of China (Grant No. 30560097, 30760156), Hi-tech Tamura K., Dudley J., Nei M., Kumar S. (2007). MEGA4: Molecular Research and Development Program of China (863 Program) (Grant No. 2006AA10Z345 and 2007AA10Z353) and New Century Evolutionary Genetics Analysis (MEGA) software version 4.0. Excellent Talent (NCET) Planning of Education Ministry of China. Mol. Biol. Evol., 24: 1596-1599. Ann. Microbiol., 59 (4), 727-731 (2009) 731 Vuilleumier S. (1997). Bacterial glutathione S-transferases: superoxide dismutase activity. Biochim. Biophys. Acta., 1780: 869-872. what are they good for? J. Bacteriol., 179: 1431-1441. Zhang W., Yu D., Sun Z., Chen X., Bao Q., Meng H., Hu S., Zhang H. Vuilleumier S., Pagni M. (2002). The elusive roles of bacterial (2008a). Complete nucleotide sequence of plasmid plca36 iso- glutathione S-transferases: new lessons from genomes. Appl. lated from Lactobacillus casei Zhang. Plasmid., 60: 131-135. Microbiol., 58: 138-146. Zhang W., Yun Y., Sun T., Menghe B., Zhang H. (2008b). Isolation Yan F., Yang W.K., Li X.Y., Lin T.T., Lun Y.N., Lin F., Lv S.W., Yan and identification of dominant microorganisms involved in G.L., Liu J.Q., Shen J.C., et al. (2008). A trifunctional enzyme naturally fermented goat milk in Haixi region of Qinghai, with glutathione S-transferase, glutathione peroxidase and China. Ann. Microbiol., 58: 213-217. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Preliminary analysis of glutathione S-transferase homolog fromLactobacillus casei Zhang

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Springer Journals
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Copyright © 2009 by University of Milan and Springer
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 59 (4) 727-731 (2009) Preliminary analysis of glutathione S-transferase homolog from Lactobacillus casei Zhang 1§ 2,3§ 1 1 2,3 4 Wen Yi ZHANG , Dong Liang YU , Zhi Hong SUN , Caicike AIRIDENG , Song Nian HU , He MENG *, He Ping ZHANG * Key Laboratory of Dairy Biotechnology and Engineering Ministry of Education, College of Food Science and Engineering, Inner Mongolia Agricultural University, Huhhot 010018; Key Laboratory of Genome Science and Information, Beijing In- stitute of Genomics, Chinese Academy of Sciences, Beijing 100029; James D. Watson Institute of Genome Sciences, Zhe- jiang University, Hangzhou 310029; School of Agriculture and biology, Shanghai Jiao Tong University, Shanghai 200240, China Received 12 June 2009 / Accepted 28 September 2009 Abstract - Glutathione S-transferases (GSTs) constitute a large protein family. Some of them are proved to be associated with biphenyl metabolism. In this study, we described a homolog of GST gene in Lactobacillus casei Zhang that revealed by genome analysis. The GST gene itself as well as the adjacent gene context was highly conserved among different L. casei isolates. Phylogenetic investigation suggested that the GST gene was acquired via horizontal gene transfer event, possibly from gamma subdivision. Additional survival experiment indicated that it may not benefit the L. casei host for growing in the presence of biphenyl. It was for the first time discovered that GST was encoded by Lactobacillaceae species. Key words: Lactobacillus casei Zhang; glutathione S-transferase; adjacent genes; horizontal gene transfer. INTRODUCTION of China (Zhang et al., 2008a). Attempting to track its potential biological role, distribution, phylogenetic, and preliminary func- Bacterial glutathione S-transferases (GSTs, EC 2.5.1.18) con- tion analysis were performed. stitute a large protein family with functional versatility as which in eukaryotic organisms, such as association with aromatic compounds metabolism and response to oxidative stress (Lloyd- MATERIALS AND METHODS Jones and Lau, 1997; Fortin et al., 2006). Hitherto they have been broadly identified and characterized in bacterial strains, and Bacterial strains and culture conditions. Lactobacillus casei grouped into four main classes (Allocati et al., 2008). Several Zhang and other fifty-eight Lactobacillus casei isolates were studies have been carried out to assess the potentials of the GSTs obtained from Key Laboratory of Dairy Biotechnology and using both proteins and microorganisms engineering (Kurtovic Engineering Ministry of Education, Inner Mongolia Agricultural et al., 2008; Yan et al., 2008). Nevertheless, experimental and University, China. They were all identified by conventional and biotechnological investigations appeared to be undertaken within 16S rRNA methods (data unpublished). Lactobacillus casei a set of bacterial species. Firmicutes strains encoded GSTs have strains were propagated at 37 °C in Trypticase peptone yeast not been described commonly as which in Proteobacteria or broth (TPY; DIFCO, PQ, Canada). Cyanobacteria (Vuilleumier, 1997). GSTs encoded by strains of Streptococcaceae and Carnobacteriaceae have been identified, PCR amplification and sequencing. Total genomic DNA was yet did not any from Lactobacillaceae (Vuilleumier and Pagni, extracted by using Bacterial DNA isolation kit (Tiangen, Beijing, 2002). China) according to manufacturer’s instructions. Based on the In the present study, we described a homolog of GST gene finished genome sequences of L. casei Zhang, a primer pair identified in the genome of Lactobacillus casei Zhang, a new pro- (GST_F, GST_R) that designed within the GST gene was used biotic strain isolated from home-made koumiss in Inner Monglia to detect its existence. Two primer pairs (GST_L3F, GST_L3R; GST_L4F, GST_L4R) that placed in the upstream genes, encoding a saccharopine dehydrogenase related protein and a transcrip- tion regulator, were used to investigate its adjacent gene context These authors contributed equally to this work. among different L. casei isolates. Primers are listed in Table 1. * Corresponding Authors. H.P. Zhang - Phone: 86-471-4319940; PCR reaction was carried out in a 20 μl reaction mixture con- Fax: 86-471-4300122; E-mail: hepingdd@vip.sina.com; taining 0.2 μl Taq polymerase (5 U/μl, Takara Tokyo, Japan), 2.5 H. Meng - Phone: 86-21-34206145; Fax: 86-21-34204538; 2+ μl 10X PCR Buffer (Mg free), 2 μl dNTPs (2.5 mM each), 2 μl E-mail: menghe@sjtu.edu.cn 728 W.Y. ZHANG et al. TABLE 1 - PCR primers used package with neighbor-joining method (Tamura et al., 2007). Reliability of the nodes was tested with 1000 repetitions. Primer Sequence (5′-3′) Microbial counts in the presence of biphenyl. Defined GST_F TACGATCAAATCAACGCCA medium containing 10 g proteose peptone, 5 g tryptone, 3 g GST_R CGCAGAACTTCCTGCACAC yeast extract, 1 g Tween-80, 5 ml solution B (10 g MgSO ·7H O, 4 2 0.5 g FeSO ·7H O, 0.5 g NaCl, 0.337 g MnSO , 250 ml ddH O), GST_L3F ACAATTAGGAAAAGAACACGG 4 2 4 2 0.2 g L-cyctein HCl, and 1000 ml Bacto-Liver extract was used GST_L3R GCTCGAAAATGATACGACACT for strain cultivation (Kozaki et al., 1992). Overnight cultures of L. casei Zhang were inoculated in the defined medium and the GST_L4F GTTAGTGACTGCCATCGTGAGA defined medium supplemented with 0.5 mM biphenyl respec- GST_L4R AAGGCGAAATAAAGTTTTTGTG tively (Bartels et al., 1999). After incubation at 37 °C for 0, 12, 36, and 48 h, survivors were enumerated on TPY agar plates in triplicate (Zhang et al., 2008b). MgCl (25 mM), 1 μl forward primer (1 pM), 1 μl reverse primer (1 pM), 1 μl genomic DNA (100 ng/μl), and 10.3 μl ddH O. Nucleotide sequence accession numbers. The sequence of Amplifications were performed on a thermal cycler (Bio-Rad, L. casei Zhang GST gene is contained in its genome sequences, California, USA) as following program: 94 °C for 5 min, 94 °C for accession number CP001084. Ten partial sequences of GST gene 30 s, 56.5 °C for 30 s, 72 °C for 40 s, 45 cycles, then 72 °C for and corresponding adjacent genes of L. casei strains have been 10 min, 4 °C for 30 min. deposited in GenBank under the accession numbers of FJ750469- The PCR products were separated by agarose gel electro- FJ750478. phoresis, extracted using a Huashun Gel Extraction Kit (Huashun, Shanghai, China) and directly sequenced. Fragment sequences were assembled to contigs with EditSeq program of DNAstar RESULTS AND DISCUSSION software (DNASTAR, Madison, WI, US). PCR amplification and sequencing Analysis of the sequences. Domain characterization was Analysis of L. casei Zhang genome has identified a homolog carried out by using Interpro database (Mulder et al., 2005). of GST gene with a full-length of 630 bp (LCAZH_1208). GST Homolog searching was performed against nr database provided N-terminal (PF02798) and C-terminal (PF00043) domains were by NCBI website using BLASTP (Altschul et al., 1997), and the found in its deduced protein sequence. To further explore the most similar sequences were selected for phylogenetic analysis conservation properties of the GST sequences distributed in with the cutoff E-value 1e-3 and the identity 25%. Jalview 2.3 genus L. casei, we did the screening tests among different iso- was used to remove the redundancy of the sequences (Clamp lates by using standard PCR. Upstream regions of the GST gene et al., 2004). Phylogenetic tree was reconstructed using MEGA were determined and sequenced as well. FIG. 1 - Electrophoretic characterization of PCR amplification products of GSTs from fifty-nine different Lactobacillus casei isolates. M: DL2000 maker; lane 1-59: L. casei strain Zhang, 1301, 1302, 1303, 1305, 1307, 2303, 4305, 6301, 6304, 9303, 11301, 13303, 14302, 14303, 14306, 15301, 15302, 15304, 15305, 15306, 23302, 24304, 25302, 25303, 25304, 29301, 29303, 29305, 33304, 29307, 29306, 33303, 29308, 34303, 34304, 34306, 37302, 37303, 39305, 41303, 43301, 43302, 43306, AG8-2, AG8-3, AG8-5, AG9-1, AG9-2, AG9-4, AG9-5, XM2-1, XM7-1, ZL3-1, ZL3-2, AZ43-3-4-2, AY2-2, AZ43-3-4-1, and ZL3-3. Ann. Microbiol., 59 (4), 727-731 (2009) 729 FIG. 2 - Phylogenetic estimation of the relationship between different bacterial GSTs. Bootstrap values were reported for greater than 40% and were shown at each of the corresponding nodes. Fifty-nine isolates from five distinct regions of China were gene contexts with L. casei Zhang, a saccharopine dehydroge- employed during PCR trials. Amplifications within the DNA nase related protein and a transcription regulator were detected template of L. casei Zhang were used for control purpose. As upstream the GST. Likewise the GST detected in Escherichia coli presented in Fig. 1, except four strains (11301, 29306, 29308, K-12, it was neither part of the gene clusters of bph nor flanked and az43-3-4-2), all experimental strains represented the same by genes relevant to catabolic functions (Blattner et al., 1997). amplification pattern. Sequencing ten of the total PCR products revealed > 98% similarity with homologous regions in L. casei Phylogenetic analysis Zhang. This could indicate that the GST gene is relatively con- A gene-based tree was constructed so as to trace the origin of served among the members of genus L. casei, which suggests an GST gene identified in L. casei Zhang. Representative GST mem- important physiological role. bers from thirty-seven different bacterial species were used dur- Additional PCR reactions were performed to precisely exam- ing the phylogeny test (Fig. 2). However, owing to the low level ine gene organizations upstream the GST of previously selected of identity between different bacterial GSTs, only a few nodes are isolates. Similarly results were also obtained as analyzing the supported by high bootstrap values. The real evolution history of GST distribution. Sequences of these PCR products using prim- these GSTs is still uncertain, but the phylogenetic analysis partly ers GST_L3F and GST_L3R, or GST_L4F and GST_L4R showed > indicates the close relationship of L. casei Zhang encoded GST 96% similarity with L. casei Zhang. The ten isolates had identical gene with its counterparts from Firmicutes. 730 W.Y. ZHANG et al. FIG. 3 - Survival of Lactobacillus casei Zhang cells during the 48 h continuous cultivation: ◆ Biphenil-free, ■ Biphenil treatment. It can be observed from the main topology in the tree, most, REFERENCES if not all of the well-defined clusters are consisted of GST mem- bers from the same taxon. Among them, Escherichia coli as well Allocati N., Federici L., Masulli M., Di Ilio C. (2009). Glutathione as other five Enterobacteriales GSTs was shown as the largest transferases in bacteria. FEBS J., 276: 58-75. cluster. L. casei GST was grouped with Enterococcus faecalis Altschul S.F., Madden T.L., Schaffer A.A., Zhang J., Zhang Z., encoded GST, appeared most close to those from gamma subdi- Miller W., Lipman D.J. (1997). Gapped BLAST and PSI-BLAST: vision bacteria. Indeed, GSTs have been identified in strains from a new generation of protein database search programs. Lactobacillales, but no protein belonged to GST family has been Nucleic Acids Res., 25: 3389-3402. detected in Lactobacillaceae species. These findings indicated Bartels F., Backhaus S., Moore E.R., Timmis K.N., Hofer B. that GST encoded by L. casei might acquire from the horizontal (1999). Occurrence and expression of glutathione-S-trans- gene transfer event. ferase-encoding bphK genes in Burkholderia sp. strain LB400 and other biphenyl-utilizing bacteria. Microbiology, 145: Preliminary function assay 2821-2834. Since GST information of dairy-related bacteria has not been reported, a greedy search was performed with BLAST. Analysis Blattner F.R., Plunkett G., 3rd Bloch C.A., Perna N.T., Burland V., results indicated that GST encoded by L. casei Zhang was a novel Riley M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew gene, no homologous gene can be found in any other sequenced G.F., et al. (1997). The complete genome sequence of Lactobacillus genomes. The putative GST protein shares 37% Escherichia coli K-12. Science, 277: 1453-1474. identity to BphK encoded by Burkholderia xenovorans LB400. Clamp M., Cuff J., Searle S.M., Barton G.J. (2004). The Jalview Most often, it was found located downstream of the gene clusters Java alignment editor. Bioinformatics, 20: 426-427. encoding degradation pathway of biphenyl (bph) (Ohtsubo et al., Fortin P.D., Horsman G.P., Yang H.M., Eltis L.D. (2006). A glutath- 2001). Bartels et al. (1999) has demonstrated that BphK was ione S-transferase catalyzes the dehalogenation of inhibitory able to fulfill the function of biphenyl (BP) utilization to their host metabolites of polychlorinated biphenyls. J. Bacteriol., 188: organisms, although it was not essential. 4424-4430. To test whether the GST gene can be an advantage for L. casei Zhang in the presence of biphenyl, viability of the cells Kozaki M., Uchimura T., Okada S. (1992). Laboratory Manual of was measured. After 48 h consecutive incubation, total viable Lactic Acid Bacteria, Asakura Book Store, Tokyo. cells of L. casei Zhang in the biphenyl-free medium increased, Kurtovic S., Shokeer A., Mannervik B. (2008). Diverging catalytic which ranged from 8.83 to 9.78 log CFU/ml (Fig. 3). On the capacities and selectivity profiles with haloalkane substrates contrary, cells propagated in the biphenyl treatment medium not of chimeric alpha class glutathione transferases. Protein Eng. only presented a slow growth rate but also seemed decreased Des. Sel., 21: 329-341. at some level during the latter 36 h. Therefore, we tentatively Lloyd-Jones G., Lau P.C. (1997). Glutathione S-transferase- propose that the cells of L. casei Zhang may not have the ability encoding gene as a potential probe for environmental bacte- to utilize biphenyl. However, this does not reflect the useless role rial isolates capable of degrading polycyclic aromatic hydro- GST played in its host L. casei strain. Further studies about GST carbons. Appl. Environ. Microbiol., 63: 3286-3290. should be performed for demonstrating its biological roles in L. casei. Mulder N.J., Apweiler R., Attwood T.K., Bairoch A., Bateman In conclusion, we identified and characterized a homolog of A., Binns D., Bradley P., Bork P., Bucher P., Cerutti L., et al. GST gene encoded by L. casei Zhang genome. Although it was (2005). InterPro, progress and status in. Nucleic Acids Res., done within a preliminary frame, analysis results from distribu- 33: D201-205. tion, phylogenetic and function tests make it possible for future Ohtsubo Y., Delawary M., Kimbara K., Takagi M., Ohta A., Nagata associated works. Y. (2001). BphS, a key transcriptional regulator of bph genes involved in polychlorinated biphenyl/biphenyl degradation Acknowledgements in Pseudomonas sp. KKS102. J. Biol. Chem., 276: 36146- This research was supported by National Natural Science Foundation of China (Grant No. 30560097, 30760156), Hi-tech Tamura K., Dudley J., Nei M., Kumar S. (2007). MEGA4: Molecular Research and Development Program of China (863 Program) (Grant No. 2006AA10Z345 and 2007AA10Z353) and New Century Evolutionary Genetics Analysis (MEGA) software version 4.0. Excellent Talent (NCET) Planning of Education Ministry of China. Mol. Biol. Evol., 24: 1596-1599. Ann. Microbiol., 59 (4), 727-731 (2009) 731 Vuilleumier S. (1997). Bacterial glutathione S-transferases: superoxide dismutase activity. Biochim. Biophys. Acta., 1780: 869-872. what are they good for? J. Bacteriol., 179: 1431-1441. Zhang W., Yu D., Sun Z., Chen X., Bao Q., Meng H., Hu S., Zhang H. Vuilleumier S., Pagni M. (2002). The elusive roles of bacterial (2008a). Complete nucleotide sequence of plasmid plca36 iso- glutathione S-transferases: new lessons from genomes. Appl. lated from Lactobacillus casei Zhang. Plasmid., 60: 131-135. Microbiol., 58: 138-146. Zhang W., Yun Y., Sun T., Menghe B., Zhang H. (2008b). Isolation Yan F., Yang W.K., Li X.Y., Lin T.T., Lun Y.N., Lin F., Lv S.W., Yan and identification of dominant microorganisms involved in G.L., Liu J.Q., Shen J.C., et al. (2008). A trifunctional enzyme naturally fermented goat milk in Haixi region of Qinghai, with glutathione S-transferase, glutathione peroxidase and China. Ann. Microbiol., 58: 213-217.

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Published: Jan 5, 2010

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