Isolation and characterization of salt-sensitive mutants of the moderately halophilic bacterium Salinivibrio costicola subsp. yaniae

Salinivibrio costicola subsp. yaniae is a moderately halophilic bacterium which can grow over a wide range of salinity. In response to external osmotic stress (1-3 M NaCl), S. costicola subsp. yaniae can accumulate ectoine, glycine betaine, and glutamate as compatible solutes. We used suicide plasmids pSUP101 to introduce transposon Tn1732 into S. costicola subsp. yaniae via Escherichia coli SM10 mediated by conjugation. One Tn1732-induced mutant, MU1, which was very sensitive to the external salt concentration, was isolated. Mutant MU1 did not grow above 1.5 M NaCl and did not synthesize ectoine, but accumulated Ngamma-acetyldiaminobutyrate, an ectoine precursor, as confirmed by (1)H-NMR analysis. From these data, we concluded that ectoine performs a key role in osmotic adaptation towards high salinity environments in strain S. costicola subsp. yaniae.     

A halophilic serine proteinase from Halobacillus sp. SR5-3 isolated from fish sauce: purification and characterization

A halophilic bacterium was isolated from fish sauce, classified, and named Halobacillus sp. SR5-3. A purified 43-kDa proteinase produced by this bacterium showed optimal activity at 50 degrees C and pH 9-10 in 20% NaCl. The activity of the enzyme was enhanced about 2.5-fold by the addition of 20-35% NaCl, and the enzyme was highly stabilized by NaCl. It was found to be a serine proteinase related to either chymotrypsin or subtilisin. It absolutely preferred Ile at the P(2) position of substrates. Thus, the enzyme was found to be a halophilic serine proteinase with unique substrate specificity.     

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.     

Effect of hyperosmotic shock on phosphoenolpyruvate carboxykinase gene expression and gluconeogenic activity in the crab muscle

Chasmagnathus granulata phosphoenolpyruvate carboxykinase (PEPCK) cDNA from jaw muscle was cloned and sequenced, showing a specific domain to bind phosphoenolpyruvate in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), respectively, specific for all PEPCKs. In the kinase-1 motifs the GK was changed to RK. The first 19 amino acids of the putative enzyme contain hydrophobic amino acids and hydroxylated residues specific to a mitochondrial type signal. The PEPCK is expressed in hepatopancreas, muscles, nervous system, heart, and gills. Hyperosmotic stress for 24 h increased the PEPCK mRNA level, gluconeogenic and PEPCK activities in muscle.     

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.     

Cell-bound cations of the moderately halophilic bacterium Vibrio costicola.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.     

Structural Changes in Halophilic and Non-halophilic Proteases in Response to Chaotropic Reagents

Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein–urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca²⁺ and Na⁺ ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.     

Roles of Asp75, Asp78, and Glu83 of GTP-dependent phosphoenolpyruvate carboxykinase from Mycobacterium smegmatis

The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K(m) value for Mn(2+) 170-fold. In contrast, Asp(75) is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V(max) decreased 1.4-4-fold. K(m) values for PEP and Mn(2+) increased 3-9- and 1.2-10-fold, respectively. K(m) values for GDP and bicarbonate changed little. 2) For PEP formation, V(max) increased 1.5-2.7-fold. K(m) values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.     

Discovery of PPi-type Phosphoenolpyruvate Carboxykinase Genes in Eukaryotes and Bacteria

Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO₂ to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PP_i)-type PEPCK (PP_i-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PP_i-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PP_i-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PP_i-PEPCK activity. The primary structure of PP_i-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PP_i-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PP_i-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply.     

Cell envelope phospholipid changes in a moderate halophile during phenotypic adaptation to altered salinity and osmotic stress

Abstract The increased content of negatively-charged phospholipids in membranes of Vibrio costicola grown at high salinities is mediated by increased phospholipid synthesis of phosphatidylglycerol relative to phosphatidylethanolamine. This phenomenon provides a system for investigating the factors involved in triggering and controlling haloadaptation in this moderately halophilic bacterium. We review recent experiments, which show that when subjected to sudden increases in external salinity, V. costicola senses both the absolute NaCl concentration and the magnitude of the salt shift. We show that the latter is sensed at least in part via osmotic pressure effects, since shift-up into sucrose-containing media triggers comparable changes in growth and in phospholipid composition and synthesis.     

Structural investigation of the binding of nucleotide to phosphoenolpyruvate carboxykinase by NMR

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reversible conversion of oxalacetate and GTP to phosphoenolpyruvate (PEP), GDP, and CO2. PEPCK from higher organisms is a monomer, specifically requires GTP or ITP, and uses Mn2+ as the activating cation. Currently, there is no crystal structure of GTP-utilizing PEPCKs. The conformation of the bound nucleotide was determined from transferred nuclear Overhauser effects (trnOe) experiments to determine internuclear proton distances. At 600 MHz in the presence of PEPCK, nOe effects were observed between nucleotide protons. Internuclear distances were calculated from the initial rate of the nOe buildup. These distance constraints were used in energy minimization calculations to determine the conformation of PEPCK-bound GTP. The bound nucleotide has the base oriented anti to the C2'-endo(2E) ribose ring conformation. Relaxation rate studies indicate that there is an additional relaxation effect on the C1' proton upon nucleotide binding to PEPCK. Nucleotide binding to PEPCK-Mn2+ was studied by 1H relaxation rate studies, but results were complicated by long dipole-dipole distances and the presence of competing complexes. Modification of PEPCK by iodoacetamido-TEMPO leads to an inactive enzyme that is spin-labeled at cys273. The interaction of TEMPO-PEPCK with GTP allows for the measurement of nuclear distances between GTP and the spin label. The results suggest that cys273 lies near the ribose ring of the bound nucleotide, but it is too far to be implicated in direct hydrogen bonding interactions consistent with previous results [Makinen, A. L., and Nowak, T. J. Biol. Chem. (1989) 264, 12148], suggesting that cys273 does not actively participate in catalysis. Modification of PEPCK with several cysteine specific modifying agents causes no change in the ability of the enzyme to bind nucleotide as monitored by fluorescence quenching. A correlation between the size of the modifying agent and the maximal observed quenching upon saturation of the enzyme with nucleotide is observed. This suggests a mechanism for inactivation of PEPCK by cysteine modification due to inhibition of a dynamic motion that may occur upon nucleotide binding.     

Preparation and properties of highly-purified Vibrio costicola polynucleotide phosphorylase

Vibrio costicola polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) has been purified to electrophoretic homogeneity. It has an approximate molecular weight of 220 000 and consists of identical subunits with an approximate molecular weight of 72 000. The enzyme appears to be a fairly typical polynucleotide phosphorylase with respect to its pH optima, substrate specificity and requirement for a divalent cation cofactor. However, the effect of salt concentration on its physiologically important phosphorolysis activity suggests that it is a moderately halophilic enzyme, able to function at the intracellular ionic strength of the bacterium. In addition, its ADP polymerization activity is remarkably stimulated by polylysine.     

Haloadaptation: insights from comparative modeling studies of halophilic archaeal DHFRs

Proteins of halophilic archaea function in high-salt concentrations that inactivate or precipitate homologous proteins from non-halophilic species. Haloadaptation and the mechanism behind the phenomenon are not yet fully understood. In order to obtain useful information, homology modeling studies of dihydrofolate reductases (DHFRs) from halophilic archaea were performed that led to the construction of structural models. These models were subjected to energy minimization, structural evaluation and analysis. Complementary approaches concerning calculations of the amino acid composition and visual inspection of the surfaces and cores of the models, as well as calculations of electrostatic surface potentials, in comparison to non-halophilic DHFRs were also performed. The results provide evidence that sheds some light on the phenomenon of haloadaptation: DHFRs from halophilic archaea may maintain their fold, in high-salt concentrations, by sharing highly negatively charged surfaces and weak hydrophobic cores.     

Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.     

极端嗜盐硫解酶基因的克隆和氨基酸组成分析

根据嗜盐菌(Halobacterium salinarum)NRC\|34001中硫解酶的基因序列信息,采用PCR技术从菌株Halobacterium sp.ZP\|6中克隆了极端嗜盐硫解酶的基因,并对此酶的氨基组成进行了分析。同非嗜盐硫解酶相比,极端嗜盐硫解酶不但含有较多的负电荷氨基酸,较少的正电荷氨基酸和强疏水氨基酸,而且同类氨基酸中的小氨基酸含量明显增高。这表明极端嗜盐硫解酶的嗜盐特性不单来自形成的分子静电屏蔽网和疏水作用的调节,且与分子表面张力减小密切相关。     

Membrane-bound 5'-nucleotidase in marine luminous bacteria: biochemical and immunological properties

A novel 5'-nucleotidase previously described in halophilic Vibrio costicola was detected in marine Vibrio and Photobacterium strains. The enzyme of marine bacteria was similar in its properties to the 5'-nucleotidase of Vibrio costicola; it was outwardly oriented in the cytoplasmic membrane and dephosphorylated nucleoside 5'-tri-, di-, and mono-phosphates to respective nucleosides before uptake. The enzyme in marine strains was immunologically cross-reactive with the antibody raised against the purified 5'-nucleotidase of Vibrio costicola. The uptake of the products of ATP hydrolysis was studied in Vibrio harveyi, and it was shown that both adenosine and inorganic phosphate released upon the action of 5'-nucleotidase were rapidly taken up by the cell.     

Influence of salinity upon the phenotypic expression of antibiotic resistance in nonhalophilic and halophilic vibrios

The purpose of the present work is to demonstrate the influence of different NaCI concentrations included in the Mueller Hinton medium, upon the antibiotic susceptibility of 10 non-halophilic and 28 halophilic Vibrio strains. The highest number of resistance aspects were recorded at 1% NaCl concentration for V. cholerae O1/non O1 strains and at 3% for V. parahaemolyticus, V. algynolyticus, V. vulnificus, V. fisheri, V. anguillarum and V. metschnikovii.     

Protein Hypersaline Adaptation: Insight from Amino Acids with Machine Learning Algorithms

Traditional bioinformatics methods performed systematic comparison between the halophilic proteins and their non-halophilic homologues, to investigate the features related to hypersaline adaptation. Therefore, proposing some quantitative models to explain the sequence-characteristic relationship of halophilic proteins might shed new light on haloadaptation and help to design new biocatalysts adapt to high salt concentration. Five machine learning algorithm, including three linear and two non-linear methods were used to discriminate halophilic and their non-halophilic counterparts and the prediction accuracy was encouraging. The best prediction reliability for halophilic proteins was achieved by artificial neural network and support vector machine and reached 80 %, for non-halophilic proteins, it was achieved by linear regression and reached 100 %. Besides, the linear models have captured some clues for protein halo-stability. Among them, lower frequency of Ser in halophilic protein has not been report before.     

Halophilic enzymes: proteins with a grain of salt

Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1-4 M range, for activity and stability, and a high excess of acidic over basic amino residues. The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe-2S protein ferredoxin. It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein.