[Show abstract][Hide abstract] ABSTRACT: NhaH is a novel Na(+)/H(+)antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na(+)(Li(+))/H(+) antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na(+) and Li(+) without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na(+) and Li(+) but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H(+) binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na(+) and Li(+), indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li(+), without affecting that for Na(+), suggesting that E229 may be mainly responsible for Li(+)binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na(+) and Li(+), indicating that H87 and H88 are involved in H(+) binding or pH regulation.
[Show abstract][Hide abstract] ABSTRACT: We summarized the key handicap and troubleshooting when proteomic techniques were used to investigate extremophilic microorganisms, and the actual state of their proteomes research in recent years. Up to now, proteomics techniques keep developing and improving rapidly, but they has not been widely used to explore proteome of extremophilic microorganisms including halophiles, thermophiles, psychophiles, acidophiles and alkaliphiles due to specific problems including incomplete dissociation of protein-protein complexes of extremophiles, and a lot of proteins synthesized by extremophiles are resistant to the conditions which dissociated and denatured proteins synthesized by mesophilic organisms. However, the foreground of potential application of the techniques draws people on attempting zealously multifarious methods. At the present time, several technical problems for separating halophilic proteins, integral membrane proteins and predicting the function of new proteins have been solved availably. Proteomics data have validated some conclusions of genome predictions, and revealed many novel proteins and a few properties of extremophiles can not be resolved fully by genome data. The investigation of extremophiles proteomes indicated that a comprehensive view of protein expression profiles should rely on more than one proteomic method. In addition, the mutual verification of conclusions on the basis of genome and proteome and combination of these two techniques must accelerate the study of extremophilic microorganisms, and redound to uncover deeply and wholly the unique mechanisms of microorganisms adaptation to extreme environments. Moreover, it would clarify the mechanisms of their survival, and point out new direction of survey for improving damage result from stresses, finally contribute to human survival and healthy.
[Show abstract][Hide abstract] ABSTRACT: The Na+/H+ antiporter plays key roles in maintaining low cytoplasmic Na+ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters.
In our previous study, the first Na+/H+ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues (395PLIKKLGMI403) existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy
and designated as nhaHΔC. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly
inhibited the complementation ability of E. coli KNabc, in which three main Na+/H+ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coli KNabc/nhaHΔC decreased both Na+/H+ and Li+/H+ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na+, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and
Chinese Science Bulletin 01/2008; 53(21):3311-3316. DOI:10.1007/s11434-008-0431-1 · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: By using Southern blot hybridization and inverse polymerase chain reaction, a 5.5-kb DNA fragment was obtained from the genomic DNA of Halobacillus trueperi DSM10404(T). Sequence analysis revealed that it contained a potential operon with high levels of sequence similarity to the opuA operon encoding glycine betaine transporter from Bacillus subtilis, which is a member of the ATP-binding cassette (ABC) substrate binding the protein-dependent transporter superfamily. The potential operon, designated as qatA (quaternary amine transporter), consists of three structural genes, which are predicted to encode an ATP-binding protein (QatAA), a membrane-associated protein (QatAB), and an extracellular substrate-binding protein (QatAC). Moreover, the putative promoter region of the operon was found with close homology to the sigma(A)-dependent promoter of B. subtilis. Reverse transcription (RT)-PCR analysis revealed that qatAA, qatAB, and qatAC genes were transcribed in cells of H. trueperi. Cells of Escherichia coli mutant MKH13 harboring qatA on pAY41 were able to grow on selective M9 salt medium containing glycine betaine and accumulated glycine betaine in the cytoplasm, showing that qatAA, qatAB, and qatAC genes together encode a functional glycine betaine transporter.
Current Microbiology 03/2007; 54(2):124-30. DOI:10.1007/s00284-006-0235-y · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sinorhizobium fredii RT19 can tolerate up to 0.6 M NaCl, whereas all its pha2-disrupted mutants, constructed by Tn5 mutagenesis, failed to grow in even the presence of 0.1 M NaCl. No growth difference was detected in pha2 mutants at a pH<7.5 in the presence or absence of K+, but growth reduction was observed in the presence of K+ when pH>7.5. The pha2 gene cluster was able to completely restore the growth of the pha2 mutants of S. fredii RT19 in 0.6 M NaCl. Measurement of monovalent cation intracellular content suggested that pha2 was involved in both Na+ (Li+) and K+ efflux. The pha2 mutants exhibited K+/H+, but no apparent Na+(Li+)/H+ antiporter activity in everted membrane vesicles. Taken together, these results indicated that the pha2 cluster of S. fredii RT19 encodes a monovalent cation/proton antiporter involved in resistance to Na+ and adaption to pH, which was very different from the pha1 cluster of Sinorhizobium meliloti, which encodes a K+/H+ antiporter.
[Show abstract][Hide abstract] ABSTRACT: Sinorhizobium fredii RT19, a strain of freeliving bacteria, was subjected to salt shock and its protein expression profiles were analyzed by differential
display proteome approaches. The results of separation by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) showed
that the number of resolved proteins was 481, 465 and 424, corresponding to salt-free control, 5 and 50 min 1 mol/L salt treatment,
respectively. Among the resolved proteins, 82 in total had altered expression in response to salt-shock stress. 26 out of
the 82 proteins were induced and 23 were completely inhibited, while 12 were up-regulated and 21 down-regulated in response
to salt shock. In addition, the appearance of differentially displayed proteins responding to different salt shock periods
is also reported. The identity of the 26 induced proteins was revealed by matrix assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF/MS) followed by database searching. Among them, 20 were assigned to proteins with known functions.
Their roles in response to salt shock stress are discussed.
Chinese Science Bulletin 09/2004; 49(17):1828-1833. DOI:10.1007/BF03183409 · 1.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Halobacillus trueperi accumulates glycine betaine under condition of high osmolarity. A fragment of the glycine betaine transporter betH gene was obtained from the genome of H. trueperi with degenerate primers. Through Southern blot hybridization and inverse PCR, a 5.1 kb EcoRI fragment containing the complete betH gene was identified and subsequently sequenced. The betH gene was predicted to encode a 55.2 kDa protein (504 amino acid residues) with 12 transmembrane regions. BetH showed 56% identity to the OpuD of Bacillus subtilis which belongs to the betaine/carnitine/choline transporter (BCCT) family. Its putative promoter region was highly homologous to sigmaB-dependent promoter of B. subtilis. A 2.6 kb fragment containing the betH gene was cloned into pUC18 and transformed into the Escherichia coli MKH13. The accumulation of glycine betaine in transformed E. coli MKH13 bacteria was confirmed using 13C nuclear magnetic resonance spectroscopy.