Characteristics of single chemoreceptive units sensitive to amino acids and related substances in the crayfish leg

Summary Impulses in single afferent fibres from amino acid receptors were recorded extracellularly. Doseresponse relations were determined for different superfused amino acids; the relations all had a slope of 1, a common saturation level, and the action of different amino acids was characterized by a specific half saturation concentration,K _M. The most effective amino acids were always L-serine, L-alanine and L-histidine, having aK _M of 10^–5, 2·10^–5 and 1.5·10^–4 mol/l, respectively. The sequence of effective amino acids was the same for all units tested. Structural requirements for optimal stimulatory action of the amino acid molecules were concluded.Abbreviation vH van Harreveld solution This work was supported by the Deutsche ForschungsgemeinschaftWe gratefully acknowledge assistance in electronics from Mr. W. Zeitz, and in mechanics from Mr. D. Beyer and Mr. L. Müller. Technical help was provided by Mrs. E. Köster, secretarial help by Mrs. L. Bauer.     

A neutral proteinase produced by Bacillus cereus with high sequence homology to thermolysin: Production,isolation and characterization

Summary Extracellular neutral proteinase was produced in 10 l and 240 l batch cultivations of Bacillus isolate X-3, identified as B. cereus and deposited as DSM 3101. The enzyme concentration was about 37–47 mg/l in the fermentation broth. The enzyme was extracted from the medium by adsorption chromatography with Amberlite XAD-7-resin, and further purified by acetone precipitation and affinity chromatography. The mol. wt. is 35 000 Da. The enzyme is thermostabilized by calcium, inhibited by EDTA and o-phenanthrolin and has its pH-optimum at pH 6.8. The specific activity is 4.36·10^-4 kat·mg^-1 at 35°C and the k _cat/K _m on FAGLA (furylacryloyl-glyleu-NH₂) is 2.25·10⁴ M^-1 s^-1 at 30°C, pH 6.8. The proteinase is stable up to 60°C. The N-terminal amino acid sequence exhibits a high sequence homology (63%) to thermolysin and a low homology (18%) to B. subtilis neutral protease A. The enzyme may therefore be suitable for structural comparison with thermolysin in order to study factors affecting thermostability.     

Characterization of Recombinant Integrase of Human Immunodeficiency Virus Type 1 (Isolate Bru)

Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virusencoded integrase protein. The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene. Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli). The main kinetic parameters of HIV-1 integrase (K _m = (3.7 ± 0.2)·10^–10 M, k _cat = (1.2 ± 0.3)·10^–7 sec^–1) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate. Inhibition of integrase by aurintricarbonic acid ([I]₅₀ = 6.3 ± 0.4 M) and dependence of integrase activity on Mg²⁺ and Mn²⁺ concentration were studied.     

Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed inEscherichia coli.The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256–257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)₆tag was expressed at high levels inE. colias inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni²⁺affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256–257 to remove 12 amino acids including the (His)₆tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein inE. coli.In addition, the refolded CMV PR could be crystallized for X-ray diffraction.     

Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Optimization of ω-3 fatty acid production by microalgae: crossover effects of CO2 and light intensity under batch and continuous cultivation modes

The microalga Pavlova lutheri is a potential source of economically valuable docosahexaenoic and eicosapentaenoic acids. Specific chemical and physical culture conditions may enhance their biochemical synthesis. There are studies relating the effect of CO₂ on growth; however, this parameter should not be assessed independently, as its effect strongly depends on the light intensity available. In this research, the combined effects of light intensity and CO₂ content on growth and fatty acid profile in P. lutheri were ascertained, in order to optimize polyunsaturated fatty acid production. The influence of the operation mode was also tested via growing the cultures by batch and by continuous cultivation. Higher light intensities associated with lower dilution rates promoted increases in both cell population and weight per cell. Increased levels of CO₂ favored the total lipid content, but decreased the amounts of polyunsaturated fatty acids. Mass productivities of eicosapentaenoic acid (3.61 ± 0.04 mg · L⁻¹ · d⁻¹) and docosahexaenoic acid (1.29 ± 0.01 mg · L⁻¹ · d⁻¹) were obtained in cultures supplied with 0.5% (v/v) CO₂, at a dilution rate of 0.297 d⁻¹ and a light intensity of 120 μE · m⁻² · s⁻¹.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

RNA Affinity for Molecular L-Histidine; Genetic Code Origins

Selection for affinity for free histidine yields a single RNA aptamer, which was isolated 54 times independently. This RNA is highly specific for the side chain and binds protonated L-histidine with 10²−10³-fold stereoselectivity and a dissociation constant (K_D) of 8–54 μM in different isolates. These histidine-binding RNAs have a common internal loop–hairpin loop structure, based on a conserved RAAGUGGGKKN_0–36 AUGUN_0–2AGKAACAG sequence. Notably, the repetitively isolated sequence contains two histidine anticodons, both implicated by conservation and chemical data in amino acid affinity. This site is probably the simplest structure that can meet our histidine affinity selection, which strengthens experimental support for a “stereochemical” origin of the genetic code.[Reviewing Editor: Niles Lehman]     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Purification and Characterization of Glutamate Decarboxylase from Lactobadllus bvevis IFO 12005

Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobadllus brevis TFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH₂-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30°C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effect was as follows: ammonium sulfate?>?sodium sulfate?>?magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with l-glutamic acid as a substrate and the K_m, k_cat, and k_cat/K_m values were 9.3 mm, 6.5 s^?1, and 7 × 10² m^?1 s^?1, respectively.     

An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara

Cold acclimation in plants is a polygenic phenomenon involving increased expression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated protoplasts from warm-grown kale (Brassica oleracea) as a model system, we tested protein fractions from winter bittersweet nightshade, Solanum dulcamara, stems for the presence of proteins that have a cryoprotective effect. Purification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it has high homology to osmotin and osmotin-like proteins. When added to warm-grown protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at –8 °C. A cDNA library which was made in November from stems and leaves of S. dulcamara was successfully screened for the corresponding cDNA clone. The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues including a N-terminal signal sequence and a putative C-terminal propeptide. The mature protein, without the N-terminal signal sequence, was expressed in Escherichia coli. The partially purified protein in the supernatant fraction of the culture medium had cryoprotective activity.     

Plant Growth Retarding Activity of the 4-Substituted-7-(β-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine Anticytokinins

Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3×10^-9 M, 2.3×10^-7 M, and 3.1×10^-7 M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P₁) was found to be Arg¹⁷ by limited proteolysis by trypsin. The second reactive site (P₁) was estimated to be Leu⁴⁶, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.     

Peptide Sequencing by Mass Spectrometry for Homology Searches and Cloning of Genes

It is now possible to obtain sequence information from gel-separated proteins by mass spectrometry at levels too low for conventional approaches. Usually this tandem mass spectrometric data are used for database searches with the aim of identifying the corresponding gene. Recently it has been shown that long and accurate amino acid sequences can be obtained which are sufficient for PCR-based strategies to clone the corresponding gene [Wilm et al. (1996), Nature 379, 466–469]. More than eight proteins have now been cloned based on that method. In many more cases the sequence information identified homologous proteins. Issues involved in cloning by mass spectrometric sequence information are discussed, as are two case studies. These results clearly establish mass spectrometry as a viable tool not only for the database identification of proteins, but also for the de novo sequencing of gel-separated proteins at the low-picomole to femtomole level.     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Influence of water activity on Streptococcus diacetylactis metabolism

During the cheese-making process, water activity (a_w) is one of the essential environmental parameters acting on bacterial growth and metabolic pathways. The influence of a_w on Streptococcus diacetylactis growth and lactic acid production was studied. The specific growth rate was linearly related to water availability in the milk medium. The cell behaviour was quite different above and below a_w=0.95, which can be considered a limiting value. Below this value, the lactic acid production reached 1.4–6.1 mg·g^–1, whereas the specific productivity was 2.0–2.6 mg·10^–10 cells·h^–1. Changes in the consumption of lactose and amino acids during the different growth phases was completely modified by decreasing the water availability in the medium. Correspondence to: N. Cochet     

Olfactory sensitivity to food odor components in the short-tailed fruit bat,Carollia perspicillata (phyllostomatidae,chiroptera)

Summary The absolute olfactory sensitivity in a frui-teating bat, Carollia perspicillata, was investigated. Eighteen monomolecular food odor components from 3 substance classes were tested using a sniff rate analysis method. Detection thresholds (Table 1) ranged from 3.6 · 10¹³ to 2.7 · 10¹⁰ molecules/cm³ air. Interindividual variation (N = 4) for a substance did not exceed one order of magnitude. Significant correlations between olfactory performance and carbon chain length of the odor molecule were found for two substance classes: Sensitivity to the aliphatic iso-alcohols increased linearly from C₂ to C₅, and a nonlinear correlation was found for the acetic esters, with the C₄- and C₇-forms being clearly better perceived than the other homologues. In acetic esters, the sensitivity for the n-forms of the molecule was significantly higher than for the iso-forms. No such correlation between stereo-isomers and olfactory perception was found for the n- and iso-forms of carbon acids and aliphatic alcohols. Fruit-typical odor components like ethyl butyrate (5.4 · 10¹⁰), n-pentyl acetate (2.8 · 10¹⁰), or linalool (1.8 · 10¹¹ molecules/cm³ air) were the most effective among all compounds tested, suggesting that the nutritional specialization of the bat may be associated with a specific spectrum of olfactory sensitivity.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05

A halostable cellulase with a molecular mass of 29 kDa was purified from culture supernatants of the halophilic bacterium Salinivibrio sp. NTU-05 by way of the Fast Protein Liquid Chromatography method and the biochemical properties of the halostable cellulase was studied. The enzyme was active over a range of 0–25% sodium chloride examined in culture broth. The optimum cellulase activity was observed at 5% sodium chloride. Results from the salinity stability test indicated 24% of enzyme activity was retained at 25% sodium chloride for 4 h. The enzyme was also shown to be slightly thermostable with 40% residual activity under 60 °C for 4 h. The enzyme has a K_m of 3.03 mg/ml and a V_max of 142.86 mol/min/mg when tested using carboxymethyl-cellulose (CMC). The enzyme activity increased in the presence of K⁺, Mg²⁺, Na⁺ ions and decreased when Hg²⁺ ions were present. The deduced internal amino acid sequence of the Salinivibrio sp. NTU-05 cellulase showed similarity to the sequence of the glycoside hydrolase family protein. These are some of the novel characteristics that make this enzyme have potential applications in cellulose biodegradation.     

Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

Functional Analysis of the Superfamily 1 DNA Helicases Encoded by Mycoplasma pneumoniae and Mycoplasma genitalium

The DNA recombination and repair machinery of Mycoplasma pneumoniae is composed of a limited set of approximately 11 proteins. Two of these proteins were predicted to be encoded by neighboring open reading frames (ORFs) MPN340 and MPN341. Both ORFs were found to have sequence similarity with genes that encode proteins belonging to the DNA helicase superfamily 1 (SF1). Interestingly, while a homolog of the MPN341 ORF is present in the genome of Mycoplasma genitalium (ORF MG244), MPN340 is an M. pneumoniae-specific ORF that is not found in other mycoplasmas. Moreover, the length of MPN340 (1590 base pairs [bp]) is considerably shorter than that of MPN341 (2148 bp). Examination of the MPN340-encoded amino acid sequence indicated that it may lack a so-called 2B subdomain, which is found in most SF1 DNA helicases. Also, the MPN340-encoded amino acid sequence was found to differ between subtype 1 strain M129 and subtype 2 strain FH at three amino acid positions. Both protein variants, which were termed PcrA^s _M129 and PcrA^s _FH, respectively, as well as the MPN341- and MG244-encoded proteins (PcrA_Mpn and PcrA_Mge, respectively), were purified, and tested for their ability to interact with DNA. While PcrA_Mpn and PcrA_Mge were found to bind preferentially to single-stranded DNA, both PcrA^s _M129 and PcrA^s _FH did not demonstrate significant DNA binding. However, all four proteins were found to have divalent cation- and ATP-dependent DNA helicase activity. The proteins displayed highest activity on partially double-stranded DNA substrates carrying 3′ single-stranded extensions.