Molecular cloning and sequencing of two phospho-beta-galactosidase I and II genes of Lactobacillus gasseri JCM1031 isolated from human intestine

Lactobacillus (Lb.) gasseri JCM1031, which is classified into the B1 subgroup of the Lb. acidophilus group of lactic acid bacteria, characteristically produces two different phospho-beta-galactosidases (P-beta-gal) I and II in the same cytosol as reported in our previous papers [Biosci. Biotech. Biochem., 60, 139-141, 708-710 (1996)]. To clarify the functional and genetic properties of the two enzymes, the structural genes of P-beta-gal I and II were cloned and sequenced. The structural gene of P-beta-gal I had 1,446 bp, encoding a polypeptide of 482 amino acid residues. The structural gene of P-beta-gal II had 1,473 bp, encoding a polypeptide of 491 amino acid residues. The deduced relative molecular masses of 55,188 and 56,243 agreed well with the previous value obtained from the purified P-beta-gal I and II protein, respectively. Multiple alignment of the protein sequence of P-beta-gal I and II with those of P-beta-gals from 5 microorganisms had 30-35% identity on the amino acid level, but those with phospho-beta-glucosidases from 5 microorganisms had the relatively high identity of about 50%. Considering that this strain grows on lactose medium and shows no beta-galactosidase activity, and that purified P-beta-gal I and II can obviously hydrolyze o-nitrophenyl-beta-D-galactopyranoside 6-phosphate (substrate), and also the conservation of a cysteine residue in the molecule, the P-beta-gal I and II were each confirmed as a novel P-beta-gal enzyme.     

Characterization of an RNA-binding domain in the bacteriophage phi 29 connector

The connector of bacteriophage phi 29 is known to promote the viral prohead assembly, to bind DNA, and to drive DNA packaging into preformed viral shells in an RNA-dependent process. In this report, the phi 29 connector protein, p10, is shown to bind RNA in a sequence-independent fashion, and to possess an RNA recognition motif comprised approximately the region between residues 21 and 94 of the p10 sequence. Substitution mutants in specific amino acids of the RNA-binding domain obtained by site-directed mutagenesis showed that amino acids Phe23, His57, Phe59, and Tyr61 are critical for RNA binding and, subsequently, for DNA packaging into proheads. Proteolytic modified forms of the phi 29 connector have allowed us to conclude that the DNA- and RNA-binding domains are separated within the p10 sequence. It is also shown that RNA is stably associated to DNA-filled proheads during the DNA-packaging process.     

Inactivation of the enveloped bacteriophage phi6 by butylated hydroxytoluene and butylated hydroxyanisole

The activity of adenosine desaminase AMP-desaminase and glutaminase was studied in blood serum and the liver tissue at the normal state and with acute, subacute experimental affection of the liver as well as with the CCl4-induced liver cirrhosis and obturation jaundice in different periods. It is shown that the degree and trend of changes in the enzyme activity in the affected liver depend on the character and duration of the affecting agents action. The acute and subacute affection of the liver and one-day obturation jaundice are accompanied by a decrease in the activity of all the studied enzymes in the liver tissue. With cirrhosis induced by a multiple administration of CCl4 for three months or with a month obturation jaundice the activity of glutaminase in the liver tissue lowers and that of adenosine desaminase and AMP-desaminase increased sharply. In blood serum the activity of adenosine desaminase and AMP-desaminase increases more considerably with acute affection and cirrhosis, especially with billar one.     

Stoichiometric packaging of the three genomic segments of double-stranded RNA bacteriophage phi6

A model that explains the stoichiometric packaging of the chromosomes of phi6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.     

Primary structure of the canine adenovirus PVII protein: functional implications

The DNA sequence of an open reading frame (ORF) corresponding to the canine adenovirus type 1 (Can 1, Utrecht strain) pro-VII (PVII) protein gene was determined. 560 base pairs were sequenced from the upstream end of the HindIII-A genomic fragment. The sequence gives the 33 C-terminal residues of the penton base protein followed by the 132 residue PVII protein, thus conforming to the same order and location of these genes as in Ad2. The authenticity of this putative PVII-ORF was confirmed by N-terminal sequencing of the cleavage product VII isolated from Can1 virions and also by PCR analysis of Can1 DNA from infected cells and from virions. In addition, the sizes of PVII and VII obtained from translation of the sequence and the proteins observed on the gels were also in agreement. The PVII proteins of Can1 and Ad2 share 57% sequence homology, particularly near the N-terminal third of the molecule, including conservation of the consensus proteinase cleavage site at residue 23. The PVII protein has a predicted MW of 14,626 Da, and an pI of 12.48. Though data bank searches for homology were negative, these viral proteins have significant functional and sequence analogy with histone H3.     

Mutational analysis of the role of nucleoside triphosphatase P4 in the assembly of the RNA polymerase complex of bacteriophage phi6

Bacteriophage phi6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the Reoviridae. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.     

Antimutagenicity and the influence of physical factors in binding Lactobacillus gasseri and Bifidobacterium longum cells to amino acid pyrolysates

Antimutagenic and binding properties of 28 strains of Lactobacillus gasseri and 2 strains of Bifidobacterium longum on the mutagenicity of amino acid pyrolysates were investigated in vitro using a streptomycin-dependent (SD510) strain of Salmonella typhimurium TA 98. Four strains of L. acidophilus (SBT0274, SBT1703, SBT10239, and SBT10241) and 1 strain of B. longum (SBT 2928) exhibited the highest percentage of antimutagenicity and binding. These 5 strains were further optimized for other physical factors influencing the mechanism of binding, such as cell and mutagen concentration, pH, and incubation time. In all of the selected strains, 2 mg of cells bound with 88 to 95% of 0.2 mg of 3-amino-1,4 dimethyl-5H-pyrido[4,3-b]indole in 30 min at pH 7.0. Other amino acid pyrolysates, such as 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldi-pyrido[1,2-a:3',2'-d]imidazole, 2-amino-3-methyl-imidazo[4,5,f]quinoline, and 2-amino-3,4-dimethyl-imidazo[4,5,f]quinoline were also tested for the binding ability of these strains. We observed that the complexity of the mutagens greatly influenced the binding properties. The binding of 3-amino-1,4 dimethyl-5H-pyrido[4,3-b]indole to the purified cell walls was very high compared with that of the crude cell wall, peptidoglycan, or the cell extract. Binding was inhibited when the cell walls were subjected to treatment with metaperiodate or trichloroacetic acid but not when they were subjected to treatment with lysozyme, trypsin, or proteinase K, reflecting the role of the carbohydrate component as a binding site.     

Mapping the functional domains of bacteriophage lambda integrase protein

Bacteriophage lambda encodes a site-specific recombination system that promotes the movement of the phage genome into and out of the host bacterial chromosome. The phage-encoded integrase (Int) is composed of 356 amino acid residues and carries out the required strand exchanges by means of a type I topoisomerase activity. Int also contains two distinct DNA-binding domains that interact with two different, specific sequences (arm-type and core-type sites) on DNA. In order to help understand the mechanism of site-specific recombination, we have used a genetic approach to isolate mutants defective in different steps in the recombination reaction. We developed a genetic screen for Int mutants that are defective in catalyzing excisive recombination in vivo. These mutants were screened for proficiency in binding to the P'123 arm-type sites using the bacteriophage P22 challenge-phage assays. In all, 78 such mutants were isolated and the mutational changes mapped and sequenced. These mutants have been further characterized (1) for their ability to bind the P'1 and P'123 arm-type sites and for their ability to form the attL complex in vivo, (2) for negative dominance in vitro, (3) for the presence of type I topoisomerase activity, and (4) for the ability to resolve artificially constructed recombination intermediates. We found that (1) residues in a stretch of 88 amino acids in the middle of the protein may be involved in Int-Int interactions, (2) a region around Arg212 is involved in the catalytic site, (3) residues near the carboxyl terminus play a role in enhancing Int binding to its arm-type sites, possibly by interacting with the small amino-terminal region that has been shown to be responsible for specific recognition of the arm-type sites, and (4) residues at the very carboxyl end of the protein may be involved in modulating the cleavage or religation activities of the Int protein.     

Cloning, sequence analysis, and characterization of the genes involved in isoprimeverose metabolism in Lactobacillus pentosus

Two genes, xylP and xylQ, from the xylose regulon of Lactobacillus pentosus were cloned and sequenced. Together with the repressor gene of the regulon, xylR, the xylPQ genes form an operon which is inducible by xylose and which is transcribed from a promoter located 145 bp upstream of xylP. A putative xylR binding site (xylO) and a cre-like element, mediating CcpA-dependent catabolite repression, were found in the promoter region. L. pentosus mutants in which both xylP and xylQ (LPE1) or only xylQ (LPE2) was inactivated retained the ability to ferment xylose but were impaired in their ability to ferment isoprimeverose (alpha-D-xylopyranosyl-(1,6)-D-glucopyranose). Disruption of xylQ resulted specifically in the loss of a membrane-associated alpha-xylosidase activity when LPE1 or LPE2 cells were grown on xylose. In the membrane fraction of wild-type bacteria, alpha-xylosidase could catalyze the hydrolysis of isoprimeverose and p-nitrophenyl-alpha-D-xylopyranoside with apparent Km and Vmax values of 0.2 mM and 446 nmol/min/mg of protein, and 1.3 mM and 54 nmol/min/mg of protein, respectively. The enzyme could also hydrolyze the alpha-xylosidic linkage in xyloglucan oligosaccharides, but neither methyl-alpha-D-xylopyranoside nor alpha-glucosides were substrates. Glucose repressed the synthesis of alpha-xylosidase fivefold, and 80% of this repression was released in an L. pentosus delta ccpA mutant. The alpha-xylosidase gene was also expressed in the absence of xylose when xylR was disrupted.     

Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.     

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.     

Physical structure of the replication origin of bacteriophage lambda

The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.     

The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria

We present three experiments which serve to identify carbon and proton sidechain resonances in 13C-labeled proteins. The first is an improvement on the previously published H(C)CH-COSY experiment and comprises the application of gradients for coherence selection and a reduction in the phase cycle. The second experiment is a new (H)CCH-COSY with two carbon dimensions. The (H)CCH-COSY presents several advantages over the H(C)CH-COSY experiment in terms of better sensitivity, improved resolution and easier identification of amino acid spins systems. The third experiment is a 2D proton-edited (H)C(C)H-COSY that allows suppression of methylene resonances. All three HCCH-COSY experiments show good sensitivity and excellent solvent suppression. The 2D version can be acquired in as little as 45 minutes and the 3D versions acquired overnight. The experiments are demonstrated on a 13C-labeled sample of the second PDZ domain from human phosphatase PTP1E in H2O solution.     

Efficiency and frequency of translational coupling between the bacteriophage T4 clamp loader genes

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the "sliding clamp" of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the "clamp loader" activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes 44 and 62, which are directly adjacent on polycistronic mRNA molecules, are-to some degree-translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene 44. Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of approximately 85% and a coupling frequency of approximately 25% between the 44-62 gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.     

Chromatin structure and the expression of cardiac genes

Three experiments investigated visual search for singleton feature targets. The critical dimension on which the target differed from the nontargets was either known in advance or unknown--that is, the critical difference varied either within a dimension or across dimensions. Previous work (Treisman, 1988) had shown that, while the search reaction time (RT) functions were flat in both conditions, there was an intercept cost for the cross-dimension condition. Experiment 1 examined whether this cost would disappear when responses could be based on the detection of any (target-nontarget) difference in the display (by requiring a "heterogeneity/homogeneity" decision). The cost remained. This argues that pop-out requires (or involves) knowledge of the particular dimension in which an odd-one-out target differs from the nontargets; furthermore, that knowledge is acquired through the elimination of dimensions not containing a target. In Experiment 2, the subjects had to eliminate (or ignore) one potential source of difference in order to give a positive response (displays could contain a "noncritical" difference requiring a negative response). The result was a comparatively large cost in the within-dimension (positive) condition. This can be taken to indicate that pop-out as such does not make available information as to the particular feature value in which the target differs from the nontargets. Experiment 3 examined whether search priorities can be biased in accordance with advance knowledge of the likely source of difference. The subjects were found to have a high degree of top-down control over what particular dimension to assign priority of checking to. The implication of the results for models of visual search and selection are discussed.     

The structure of the exopolysaccharide produced by Lactobacillus helveticus 766

The exopolysaccharide produced by Lactobacillus helveticus 766 in skimmed milk was found to be composed of D-glucose and D-galactose in a molar ratio of 2:1. Linkage analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and on oligosaccharides obtained from a partial acid hydrolysate, showed the polysaccharide to consist of hexasaccharide repeating units with the following structure: [formula: see text]