Molecular Cloning and Characterisation of Alpha Subunit of H + -ATPase in Lactobacillus casei Zhang

Keywords: H + -ATPase, clone, sequence, Lactobacillus casei Zhang, phylogenic trees

The acid tolerance is an important property of Lactic acid bacteria as potential probiotics. H + -ATPase is considered a key gene in several bacteria with the ability of acid tolerance. We cloned and sequenced the full length cDNA of alpha subunit of H + -ATPase gene in Lactobacillus casei Zhang, which had been isolated from traditional home-made koumiss in Inner Mongolia of China. The results showed that the respective cDNA sequence is composed of 1530 nucleotides and codes a putative protein including 509 amino acids. In addition, we also reconstructed the phylogenic trees for H + -ATPase gene based on amino acids sequences of diverse strains of Lactic acid bacteria. 

Lactic acid bacteria and other probiotic microorganisms have many documented health effects, so some of them are considered as probiotics which are defined as live microbial food supplements that benefit the health of consumers by maintaining or improving their intestinal microbial balance (Salminen et al. 1996;Fuller 1989). Moreover, the research on lactobacilli becomes more and more interesting because of their possible role in the maintenance of gastrointestinal health (Bengmark et al. 1998). Lactobacillus casei Zhang is a novel potential probiotic which was isolated from the traditional Koumiss widely used in traditional Mongolian medicine in Inner Mongolia of China (Zhang et al. 2006). This strain exhibits favourable probiotic properties such as acid tolerance, bile resistance, cholesterol-removing ability and GI colonisation ability (Wu et al. 2005;Xu et al. 2006;Zhang et al. 2006). Moreover, Yun found hypocholesterolemic effect in this strain resulting from its ability to bind and assimilate cholesterol and to suppress the reabsorption of bile acids into the enterohepatic circulation (Yun et al. 2006). Zhang found with this strain the regulating function of the cell immunity, the humoural immunity, and the intestinal mucous local immunity to mouse (Zhang et al. 2006b). 
Acid tolerance and tolerance to human gastric juice are considered as important properties in the selection of a preferable probiotic strain (Saarela et al. 2000). Studies on the physiology of oral streptococci have led to the view that the cell membrane plays major roles in acid-base regulation. These roles include the extrusion of protons through the membrane and exclusion of the environmental protons (Bender et al. 1986). Yokota et al. (1995) reported that the acid tolerance of Lactococcus lactis subsp. lactis, which is used as a starter culture in the dairy industry, depends on the action of cell membrane-bound H + -ATPase. Miwa et al. (1997) found that the H + -ATPase activity of ruminal acid-tolerant bacteria was higher than that of nonacid-tolerant bacteria, and its activity increased when the bacteria were incubated under acidic conditions. In 2000, Miwa et al. (2000) also found that H + -ATPase has a key role in the acid tolerance of Streptococcus bovis. In 2004, Matsumoto suggested that it is necessary for bacteria to increase H + -ATPase activity quickly and discharge H + in order to maintain a constant intracellular pH. The acid-intolerant strains are damaged under acidic conditions since their H + -ATPase activity cannot be increased, while acid-tolerant strains are less affected and more able to survive because of the rapid increase in their H + -ATPase activity (Matsumoto et al. 2004). 
In order to evaluate this strain much more clearly by genomic extent and determine the association between the H + -ATPase gene and acid tolerance in Lactobacillus casei Zhang, the α subunit of H + -ATPase in Lactobacillus casei Zhang was cloned and characterised. 

MATeriAl And MeTHodS


Strains and culture media. Lactobacillus casei

Zhang was obtained from the Key Laboratory of Dairy Biotechnology and Engineering Ministry of Education, Inner Mongolia Agricultural University, China. This strain can survive in acid conditions at pH 3.5 and is considered as a potential probiotic. Lactobacillus casei Zhang was cultivated in MRS broth without shaking at 37°C. 
RNA isolation. The total RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's recommendations from an overnight culture of Lactobacillus casei Zhang. 
The RNA concentration was then adjusted to 100 ng/µl by the Biophotometer (Eppendorf, Germany) and stored at -70°C until the use. 

RT-PCR.

Using RNA PCR Kit (AMV) Ver. 3.0 (TaKaRa, Japan), reverse transcriptions (RT) were performed with 10 µl reaction volume including 3 µl total RNA(100 ng/µl), 1 µl 10 × RT buffer, 2 µl 25 mmol/l MgCl 2 , 1 µl dNTP Mixture, 0.25 µl RNase Inhibitor (40 U/µl), 0.5 µl AMV Reverse Transcriptase XL (5 U/µl), 0.5 µl Oligo dT-Adaptor Primer (2.5 pmol/µl), and 1.75 µl RNase Free dH 2 O. The RT reaction conditions were as follows: 30°C for 10 min, 42°C for 30 min, 99°C for 5 min, and 5°C for 5 minutes. 
The primers were designed according to the sequence of H + -ATPase in Lactobacillus casei ATCC334 (GenBank accession numbers: NC_008526). The primers were as follows: forward 5'-AGCACCGTT-TCGATAAGA-3', reverse 5'-TGGTCGATGCGAT CTTGC-3'. The 25 µl PCR reaction mixture was composed of 0.2 µl Taq polymerase (5 U/µl, Takara Tokyo, Japan), 2.5 µl 10 × PCR Buffer (without Mg 2+ ), 2 µl dNTP (2.5mM each), 2 µl MgCl 2 (25mM), 0.2 µl forward primer (50pM), 0.2 µl reverse primer (50pM), 1 µl cDNA products and 17.4 µl ddH 2 O. The reaction conditions were as follows: 97°C for 5 min, 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles, and then 72°C for 10 min, 4°C for 30 minutes. 
Molecular cloning and sequencing. The PCR product of Lactobacillus casei Zhang was separated from 1% agarose gel electrophoresis using a Huashun Gel Extraction Kit (Huashun, China). The extracted PCR product was combined with pMD 18-T Vector (Takara, Japan) and cloned. The recombined vector was identified using restriction enzyme digestion with Hind III and BamH I (Takara, Japan) followed by 1% agarose gel electrophoresis, and then the vector was subsequently used for sequencing. 
Characteristic analysis. The α subunit of H + -ATPase gene sequence was entered into the Ed-itSeq program of the DNASTAR software package to search the largest open reading frame (ORF) and was further translated into amino acid sequences using the standard genetic code. 
The alignments of amino acid sequences of the cloned α subunit of H + -ATPase and other representatives of Lactic acid bacteria α subunit of H + -ATPase were used to generate phylogenic trees. Phylogenic trees were constructed utilising DNA-MAN software (Ver. 4.0). 

reSulTS


Sequence of α subunit of H + -ATPase in Lactobacillus casei Zhang

After RT-PCR and sequencing for confirmation, the cDNA sequence of α subunit of H + -ATPase was obtained. The cDNA sequence is composed of 1530 bp, and all the nucleotides are ORF. The ORF can be putatively composed of 509 amino acids and the translation start codon (ATG) and stop codon (TAA) were clear and emphasised by boxed (Figure 1). The gene sequence had already been available in GenBank (accession number EU370975). 

Homology analysis

To understand the sequence character of α subunit of H + -ATPase gene, the sequences of CDS and amino acids of some Lactobacillus α subunit of H + -ATPase were compared (Table 1). Then, the phylogenic tree was constructed by DNAMAN software, and is shown in Figure 2. From the table and figure, we can see that the cloned α subunit of H + -ATPase gene belongs to the group of Lactobacillus α subunit of H + -ATPase. The α subunit of H + -ATPase gene is conserved in Lactobacillus, and is highly conserved in Lactobacillus casei. 

diSCuSSion

We identified the full-length cDNA sequences of α subunit of H + -ATPase in Lactobacillus casei Zhang. The results were confirmed by sequencing and sequence analysis. The cDNA sequence consists of 1530 nucleotides, and all the nucleotides are ORF which yields a protein of 509 amino acids. Proton-translocation ATPase (F 1 F 0 complex) commonly synthesises ATP in the plasma membranes of bacteria, mitochondria, and chloroplasts. The complete nucleotide sequence of the ATPase genes of escherichia coli was determined (Walker et al. 1984). The genes for all eight subunits of the complex reside in a common operon and are transcribed into a single mRNA in bacteria (Gibson et al. 1978;Jones et al. 1983). A hierarchy was shown wherein pH optima for the enzymes were established for S. sanguis, S. salivarius, S. mutans, and Lb. casei, of approximately 7.5, 7.0, 6.0, and 5.0, respectively (Bender et al. 1986). The analysis of these data showed that the lower the pH at which the ATPase can function, the more competitive the organism as the end-products of metabolism build up. The central role of ATPase is also seen in the enteric bacteria, with which it was shown that the acid-tolerance response (ATR) does not occur in cells that are defective in the F-ATPase (Foster & Hall 1991). Consequently, it may be presumed that ATPase function is probably a major general component of acid tolerance in bacteria. 
Extensive information is now available on bacterial genes that encode the subunits of F-ATPase. The structure of F 1 F 0 -ATPase complexes from different sources are very similar and consist of Figure 1. Nucleotide sequence and putative amino acid sequence of α subunit of H + -ATPase in Lactobacillus casei Zhang 
1 ATGAGCATCAAGACTGAGGAAATCAGTTCTCTGATCAAAAAACAACTTGAAGGATATCAG 1 M S I K T E E I S S L I K K Q L E G Y Q 61 GACGATTTGGCGGCTGAAGAAGTCGGCACTGTGACTTACATCGGTGATGGGATCGCACGT 21 D D L A A E E V G T V T Y I G D G I A R 121 GCGACTGGGCTGGAAAATGCCATGGCCAACGAATTGCTCCAATTTAGCAACGGCTCATAC 41 A T G L E N A M A N E L L Q F S N G S Y 181 GGGATGGCGTTAAACCTTGAAACGAACGATGTCGGGATCATTATCTTAGGTGACTTCGAT 61 G M A L N L E T N D V G I I I L G D F D 241 GAGATTCGCGAAGGCGACCAAGTGAAACGCACTGGCCGAATCATGGAAGTTCCTGTCGGG 81 E I R E G D Q V K R T G I M E V P V G 301 GATGCCATGATCGGGCGAGTTGTCAATTCTTTGGGTCAGCCGGTCGACGGTTTAGGCGCG 101 D A M I G R V V N S L G Q P V D G L G A 361 ATTAAGACGGATAAAACCCGTCCGATCGAGTTTAAGGCGCCAGGTGTTATGCAACGCAAA 121 I K T D K T R P I E F K A P G V M Q R K 421 TCCGTATCAGAACCGTTACAAACCGGTTTGAAAGCGATTGATGCGCTGGTACCGATTGGC 141 S V S E P L Q T G L K A I D A L V P I G 481 CGTGGTCAGCGTGAGTTGATCATTGGTGACCGGAAAACCGGGAAAACATCAATTGCGATT 161 R G Q R E L I I G D R K T G K T S I A I 541 GATACCATTTTGAACCAAAAAGATCAAAACATGATCTGTGTGTACGTTGCGATTGGGCAA 181 D T I L N Q K D Q N M I C V Y V A I G Q 601 AAGGACAGTACGGTTCGAGCTCAAGTTGAAACGTTGAAAAAATATGGTGCGATGGATTAT 201 K D S T V R A Q V E T L K K Y G A M D Y 661 ACCATCGTGCTGACTGCTGGCCCATCTGAACCTGCACCAATGCTGTATATCGCGCCTTAT 221 T I V L T A G P S E P A P M L Y I A P Y 721 GCCGGTGCAGCGATGGGTGAAGAGTTCATGTATAACGGCAAGCACGTCTTGATCGTGTAT 241 A G A A M G E E F M Y N G K H V L I V Y 781 GATGATTTGAGCAAACAGGCAACCTCATATCGTGAGCTGTCCTTGCTGCTCCGTCGTCCG 261 D D L S K Q A T S Y R E L S L L L R R P 841 CCTGGTCGTGAAGCCTATCCTGGGGATATTTTCTATACCCACAGTCGCTTGTTGGAACGC 281 P G R E A Y P G D I F Y T H S R L L E R 901 GCTGCTAAATTGAGTGATAAACTCGGCGGCGGTTCTATGACGGCGTTGCCGGTTATTGAA 301 A A K L S D K L G G G S M T A L P V I E 961 ACTCAGGCAGGCGATATTTCCGCGTACATCCCGACTAACGTTATTTCTATCACGGATGGT 321 T Q A G D I S A Y I P T N V I S I T D G 1021 CAGATCTTCTTACAAAGCGATCTGTTCTATGCGGGTACACGTCCAGCCATTGATGCCGGT 341 Q I F L Q S D L F Y A G T R P A I D A G 1081 GCTTCTGTTTCCCGTGTTGGTGGTGATGCGCAGGTCAAGGCGATGAAGAAAGTTGCCGGG 361 A S V S R V G G D A Q V K A M K K V A G 1141 ACATTGCGGTTGGATCTGGCATCCTTCCGTGAACTGGAAGCCTTCACGCAATTTGGTTCT 381 T L R L D L A S F R E L E A F T Q F G S 1201 GATTTGGATGCAGCAACGCAAGCTAAGTTGAATCGTGGTCGCCGAACGGTTGAAGTGCTG 401 D L D A A T Q A K L N R G R R T V E V L 1261 AAGCAGCCTGTTCACAAACCGTTACCGGTTGAAAAGCAGGTTATCATTCTTTACGCATTA 421 K Q P V H K P L P V E K Q V I I L Y A L 1321 ACCCATGGCTTCCTTGATCCAATTCCGATTGAAGACATTACTCGCTTCCAAGATGAACTG 441 T H G F L D P I P I E D I T R F Q D E L 1381 TTTGATTTCTTCGATAGCAATGCAGCTGATTTGCTCAAGCAGATTCGTGACACCGGTAAT 461 F D F F D S N A A D L L K Q I R D T G N 1441 TTACCGGATACCGATAAACTTGATGCACAAATCAAGGCTTTTGCCGGCGGATTCCAAACG 481 L P D T D K L D A Q I K A F A G G F Q T 1501 AGTAAACAACTTGCTGCAGCGAAAGACTAA 501 S K Q L A A A K D * 1 ATGAGCATCAAGACTGAGGAAATCAGTTCTCTGATCAAAAAACAACTTGAAGGATATCAG 1 M S I K T E E I S S L I K K Q L E G Y Q 61 GACGATTTGGCGGCTGAAGAAGTCGGCACTGTGACTTACATCGGTGATGGGATCGCACGT 21 D D L A A E E V G T V T Y I G D G I A R 121 GCGACTGGGCTGGAAAATGCCATGGCCAACGAATTGCTCCAATTTAGCAACGGCTCATAC 41 A T G L E N A M A N E L L Q F S N G S Y 181 GGGATGGCGTTAAACCTTGAAACGAACGATGTCGGGATCATTATCTTAGGTGACTTCGAT 61 G M A L N L E T N D V G I I I L G D F D 241 GAGATTCGCGAAGGCGACCAAGTGAAACGCACTGGCCGAATCATGGAAGTTCCTGTCGGG 81 E I R E G D Q V K R T G R I M E V P V G 301 GATGCCATGATCGGGCGAGTTGTCAATTCTTTGGGTCAGCCGGTCGACGGTTTAGGCGCG 101 D A M I G R V V N S L G Q P V D G L G A 361 ATTAAGACGGATAAAACCCGTCCGATCGAGTTTAAGGCGCCAGGTGTTATGCAACGCAAA 121 I K T D K T R P I E F K A P G V M Q R K 421 TCCGTATCAGAACCGTTACAAACCGGTTTGAAAGCGATTGATGCGCTGGTACCGATTGGC 141 S V S E P L Q T G L K A I D A L V P I G 481 CGTGGTCAGCGTGAGTTGATCATTGGTGACCGGAAAACCGGGAAAACATCAATTGCGATT 161 R G Q R E L I I G D R K T G K T S I A I 541 GATACCATTTTGAACCAAAAAGATCAAAACATGATCTGTGTGTACGTTGCGATTGGGCAA 181 D T I L N Q K D Q N M I C V Y V A I G Q 601 AAGGACAGTACGGTTCGAGCTCAAGTTGAAACGTTGAAAAAATATGGTGCGATGGATTAT 201 K D S T V R A Q V E T L K K Y G A M D Y 661 ACCATCGTGCTGACTGCTGGCCCATCTGAACCTGCACCAATGCTGTATATCGCGCCTTAT 221 T I V L T A G P S E P A P M L Y I A P Y 721 GCCGGTGCAGCGATGGGTGAAGAGTTCATGTATAACGGCAAGCACGTCTTGATCGTGTAT 241 A G A A M G E E F M Y N G K H V L I V Y 781 GATGATTTGAGCAAACAGGCAACCTCATATCGTGAGCTGTCCTTGCTGCTCCGTCGTCCG 261 D D L S K Q A T S Y R E L S L L L R R P 841 CCTGGTCGTGAAGCCTATCCTGGGGATATTTTCTATACCCACAGTCGCTTGTTGGAACGC 281 P G R E A Y P G D I F Y T H S R L L E R 901 GCTGCTAAATTGAGTGATAAACTCGGCGGCGGTTCTATGACGGCGTTGCCGGTTATTGAA 301 A A K L S D K L G G G S M T A L P V I E 961 ACTCAGGCAGGCGATATTTCCGCGTACATCCCGACTAACGTTATTTCTATCACGGATGGT 321 T Q A G D I S A Y I P T N V I S I T D G 1021 CAGATCTTCTTACAAAGCGATCTGTTCTATGCGGGTACACGTCCAGCCATTGATGCCGGT 341 Q I F L Q S D L F Y A G T R P A I D A G 1081 GCTTCTGTTTCCCGTGTTGGTGGTGATGCGCAGGTCAAGGCGATGAAGAAAGTTGCCGGG 361 A S V S R V G G D A Q V K A M K K V A G 1141 ACATTGCGGTTGGATCTGGCATCCTTCCGTGAACTGGAAGCCTTCACGCAATTTGGTTCT 381 T L R L D L A S F R E L E A F T Q F G S 1201 GATTTGGATGCAGCAACGCAAGCTAAGTTGAATCGTGGTCGCCGAACGGTTGAAGTGCTG 401 D L D A A T Q A K L N R G R R T V E V L 1261 AAGCAGCCTGTTCACAAACCGTTACCGGTTGAAAAGCAGGTTATCATTCTTTACGCATTA 421 K Q P V H K P L P V E K Q V I I L Y A L 1321 ACCCATGGCTTCCTTGATCCAATTCCGATTGAAGACATTACTCGCTTCCAAGATGAACTG 441 T H G F L D P I P I E D I T R F Q D E L 1381 TTTGATTTCTTCGATAGCAATGCAGCTGATTTGCTCAAGCAGATTCGTGACACCGGTAAT 461 F D F F D S N A A D L L K Q I R D T G N 1441 TTACCGGATACCGATAAACTTGATGCACAAATCAAGGCTTTTGCCGGCGGATTCCAAACG 481 L P D T D K L D A Q I K A F A G G F Q T 1501 AGTAAACAACTTGCTGCAGCGAAAGACTAA 501 S K Q L A A A K D *
two parts: a membrane integral part, F 0 , which forms a proton channel, and a soluble part, F 1 , which contains the catalytic site for ATP hydrolysis (Koebmann et al. 2000). In general, the subunits in the cytoplasmic F 1 domain (consisting of the δ, α, γ, β, and ε subunits) showed a far higher level of homology as compared with the membrane-bound F 0 domain (consisting of the a, c, and b subunits) (Quivey et al. 2001). 
Lactobacillus casei Zhang is a natural lactobacillus that was isolated from traditional home-made koumiss (Zhang et al. 2006a), while Lactobacillus casei ATCC334 was isolated from emmental cheese. Moreover, a comparative sequence analysis of the genes encoding 16S rRNA of Lactobacillus casei ATCC334 and Lactobacillus casei Zhang had same 16S rRNA sequence with homology of 100% (Wang et al. 2008). However, when we checked the physiological and biochemical characteristics of these two strains, we found that Lb. casei ATCC334 can ferment rhamnose, melibiose, raffinose, and lactose, but Lb. casei Zhang can not. As we have described before, Lactobacillus casei Zhang is a potential probiotics, which can survive in artificial gastric juice. Moreover, the phylogenic tree can illustrate the relations between various Lactobacillus species. From the tree we can find that the sequences of α subunit of H + -ATPase of various Lactobacillus species are conserved. Moreover, Lb. acidophilus NCFM and Lb. helveticus DPC4571, Lb. gasseri ATCC33323 and Lb. johnsonii NCC533 are highly similar. Lb. casei ATCC334 proved not to have so highly conserved species. However, from the above results, we can see that α subunit of H + -ATPase in Lactobacillus casei is amazingly highly conserved, even if they come from different sources and countries. Thus the molecular cloning and characterisation of α subunit of H + -ATPase in Lactobacillus casei Zhang makes it possible for further research to identify the association of the H + -ATPase gene with acid tolerance. 



  Bengmark S., Larsson K., Molin G. (1998): Ecological control of the gastrointestinal tract. The role of probiotic flora. Gut, 42: 2-7. Bender G.R., Sutton S.V., Marquis R.E. (1986): Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. Infection and Immunity, 53: 331-338. 


Figure 2 .

 2 Figure 2. Homology tree of amino acid sequence of α subunit of H + -ATPase in some lactobacilli. The percent numbers represent identical degree 


Table 1 .

 1 Comparison of α subunit of H + -ATPase of Lactobacillus casei Zhang with those of other lactobacilli 


	Species	GenBank accession No.	Length (bp)	Nucleotide identity (%)	Amino acid identity (%)
	Lb. casei ATCC334	NC_008526	1530	99.74	100.00
	Lb. plantarum WCFS1	NC_004567	1515	71.11	78.98
	Lb. johnsonii NCC 533	NC_005362	1512	69.48	78.39
	Lb. gasseri ATCC 33323	NC_008530	1521	69.59	77.54
	Lb. helveticus DPC4571	NC_010080	1512	70.26	79.57
	Lb. acidophilus NCFM	NC_006814	1512	69.42	78.98
	Lb. brevis ATCC367	NC_008497	1542	69.71	74.66
	Lb. delbrueckii ATCC11842	NC_008054	1512	69.54	77.80
	Lb. reuteri 100-23	NZ_APZZ00000000	1530	68.69	75.64
	Lb. sakei subsp. sakei 23K	NC_007576	1536	72.34	81.02
	Lb. salivarius UCC118	NC_007929	1512	71.24	78.59

Supported by Natural Science Foundation of China (Grants No. 30560097, 30660135 and 30760156), Hi-tech Research and Development Program of China (863 Program) (Grants No. 2006AA10Z345 and 2007AA10Z353) and New Century Excellent Talent (NCET) Planning of Education Ministry of China.
Xia Chen, Mei Yang, Zhihong Sun, Wenjun Liu, Tiansong Sun, He Meng, Heping Zhang
Molecular Cloning and Characterisation of Alpha Subunit of H + -ATPase in Lactobacillus casei Zhang