Phytase activity in rabbit cecal bacteria

The presence of phytase activity was demonstrated in 26 strains of rabbit cecal bacteria. In 25 strains a low phytase activity, 0.10–0.62 μmol phosphate released per min per mg protein, was found. High activity (2.61 μmol/min per mg protein) was found in the strain PP2 identified as Enterococcus hirae. Phytase activity was cell-associated, being higher in the cell extract than in the cell walls. Extracellular phytase activity and cell-associated phosphatase activity were not detected. Phytase activity was optimal around pH 5.0, which is below the physiological cecal pH range. The K _m determined using the Lineweaver-Burk plot was 0.19 μmol/mL. Cations Fe³⁺, Cu²⁺ and Zn²⁺ at 0.5 mmol/L decreased phytase activity in sonicated cells of E. hirae by 99.4, 90.7 and 96.5 %, respectively. In contrast, Mg²⁺ increased activity by 11.0 %. Characteristics of E. hirae phytase (pH optimum, K _m, cation sensitivity) were similar to those of other bacterial phytases reported in the literature. Other bacteria with a high phytase activity may be present in the rabbit cecum but remain to be identified.     

One-step cloning system for isolation of bacterial lexA-like genes.

A system to isolate lexA-like genes of bacteria directly was developed. It is based upon the fact that the presence of a lexA(Def) mutation is lethal to SulA+ cells of Escherichia coli. This system is composed of a SulA- LexA(Def) HsdR- strain and a lexA-conditional killer vector (plasmid pUA165) carrying the wild-type sulA gene of E. coli and a polylinker in which foreign DNA may be inserted. By using this method, the lexA-like genes of Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa, and P. putida were cloned. We also found that the LexA repressor of S. typhimurium presented the highest affinity for the SOS boxes of E. coli in vivo, whereas the LexA protein of P. aeruginosa had the lowest. Likewise, all of these LexA repressors were cleaved by the activated RecA protein of E. coli after DNA damage. Furthermore, under high-stringency conditions, the lexA gene of E. coli hybridized with the lexA genes of S. typhimurium and E. carotovora but not with those of P. aeruginosa and P. putida.     

Rapid identification and cloning of bacterial transferrin and lactoferrin receptor protein genes.

The sequences of genes encoding the transferrin and lactoferrin receptor proteins from several bacterial species were analyzed for areas of identity in the predicted protein sequences. Degenerate oligonucleotide primers were designed and tested for their ability to amplify portions of the receptor genes. Primer pairs capable of amplifying products of the tbpA/lbpA or tbpB/lbpB genes from all species possessing these receptors were identified.     

Amidase activity of some bacteria

The amidase activity of bacteria possessing a high nitrilase activity was found to display the same spectrum although the bacteria may belong to different taxonomic groups,Bacillus, Bacteridium, Micrococcus, Brevibacterium. The spectrum of amidase activity, although very broad, is more restricted than that of nitrilase activity. Internal amides as well as vinyl-bound amides are not hydrolyzed.     

Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.

Production of a potent urease has been described as a trait common to all Helicobacter pylori so far isolated from humans with gastritis as well as peptic ulceration. The detection of urease activity from genes cloned from H. pylori was made possible by use of a shuttle cosmid vector, allowing replication and movement of cloned DNA sequences in either Escherichia coli or Campylobacter jejuni. With this approach, we cloned a 44-kb portion of H. pylori chromosomal DNA which did not lead to urease activity when introduced into E. coli but permitted, although temporarily, biosynthesis of the urease when transferred by conjugation to C. jejuni. The recombinant cosmid (pILL585) expressing the urease phenotype was mapped and used to subclone an 8.1-kb fragment (pILL590) able to confer the same property to C. jejuni recipient strains. By a series of deletions and subclonings, the urease genes were localized to a 4.2-kb region of DNA and were sequenced by the dideoxy method. Four open reading frames were found, encoding polypeptides with predicted molecular weights of 26,500 (ureA), 61,600 (ureB), 49,200 (ureC), and 15,000 (ureD). The predicted UreA and UreB polypeptides correspond to the two structural subunits of the urease enzyme; they exhibit a high degree of homology with the three structural subunits of Proteus mirabilis (56% exact matches) as well as with the unique structural subunit of jack bean urease (55.5% exact matches). Although the UreD-predicted polypeptide has domains relevant to transmembrane proteins, no precise role could be attributed to this polypeptide or to the UreC polypeptide, which both mapped to a DNA sequence shown to be required to confer urease activity to a C. jejuni recipient strain.     

A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction.

An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning.     

病原真菌和细菌毒性基因的分离和鉴别

本文讨论最新发展起来的病原真菌和细菌毒性基因的分离和鉴别原理和方法,涉及到基因表达分析法、基因转移方法、基因组比较法、诱变方法及其诱变子的筛选鉴定。着重讨论了这些方法的优点和局限性,评估了标记诱变法（ＳＴＭ）和限制酶介导的整合法（ＲＥＭＩ）两者相互结合在病原真菌毒性基因克隆中的应用潜力。     

Molecular cloning of chemotaxis genes and overproduction of gene products in the bacterial sensing system.

The chemotaxis genes cheR, cheB, cheY, cheZ, and tar of Salmonella typhimurium were cloned into bacteriophage lambda vectors and onto pBR322 plasmids by recombinant DNA techniques. The genes were linearly arranged in the order tar-cheR-cheB-cheY-cheZ (and were read from a promoter on the upstream side of the tar or cheR gene). However, their stoichiometries of expression were found to be 4:1:1:18:3, respectively. The overexpression of the cheY gene appeared to be a function of translational control. These five che genes were placed on a multicopy plasmid, and the gene products were overproduced in the cells, as shown by enzyme assays. The overproduction of the products of these five genes relative to those of the other che genes caused some changes in chemotactic properties, but no dramatic destruction of sensing ability.     

Conformational studies of bacterial polysaccharides. II. Optical activity of some bacterial capsular polysaccharides

The optical activity of the Klebsiella capsular polysaccharides of serotypes K1, K5, K6, K8, K11, K56, and K57 has been studied in aqueous solution. Measurements of ORD in the range 185–450 nm reveal anomalous ORD with Cotton effects near λ₀ = 195nm. The results are evaluated quantitatively according to hte Moffitt-Yang and the Drude equations. Straight lines are obtained in the Moffitt-Yang plots, while the corresponding Drude plots yield bent curves. The b₀ values, calculated from the slope of the stright lines in the Moffitt-Yang plot, range from 90 to 270 and suggest a helical superstructure for the capsular polysaccyharides. Positive b₀ values have been found for K1, K5, and K6 and negative b₀ values for K8, k11, K56, ad K57. Circular dichrosim has been mesured, but the CD curves are found to be truncated at the lower-wavelength end due to the 185-nm limit of the spectrometer used. Measurements of the temperature dependence of the specific optical rotation [α] reveal in all cases cooperative order–disorder transitions at temperatures, T_m, fro m298 to 323°K. The van′t Hoff enthalpies derived from the width of the transition curves are found to be similar in value to those of polypeptieds in aqueous solution. The K8 polysaccharide shows a two-step transition. The results are discussed in relation to the known primary structure and x-ray data from oriented and partially crystalline films. A model is suggested for the two-step transition in the K8 polysaccharide.     

Search for alternative secondary structures of RNA, regulating expression of bacterial genes

Expression of many bacterial genes is regulated by formation of alternative secondary RNA structure within the leader mRNA sequence. Our algorithm designed to search for these structures (basing on analysis of one nucleotide sequence) was applied to analyze operons of amino acid biosynthesis in alpha- and gamma-proteobacteria. The attenuators of these operons are predicted for genomes of some poorly known gamma-proteobacteria including Shewanella putrefaciens, attenuators of the tryptophan operon in some alpha-proteobacteria are also predicted.     

Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.

A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).     

Characterization of aquatic bacteria and cloning of genes specifying partial degradation of 2,4-dichlorophenoxyacetic acid.

Water samples from rivers, streams, ponds, and activated sewage were tested for the presence of bacteria which utilize 2,4-dichlorophenoxyacetic acid (2,4-D) as a sole source of carbon. Seventy percent of the attempted enrichments yielded pure cultures of 2,4-D-metabolizing bacteria. All but 1 of the 30 isolates were gram-negative rods, all but 2 were motile, and all were nonfermentative and oxidase and catalase positive. Nine isolates had DNA guanine-plus-cytosine values of 61.1 to 65 mol%. One isolate had a 67 mol% guanine-plus-cytosine value. The results suggest that these 2,4-D-metabolizing bacteria belong to the genus Alcaligenes. Fourteen of 23 isolates contained one or more detectable plasmids of about 20, 60, or 100 megadaltons. HindIII restriction fragment patterns showed these plasmids to be different from each other with one exception. Very similar restriction fragment patterns were revealed with a plasmid isolated from an Alcaligenes eutrophus strain obtained from Australia (pJMP397) and in an Alcaligenes sp. isolated in Oregon (pEML159). These two plasmids were about 56 megadaltons, had the same guanine-plus-cytosine value, were transmissable, and coded for 2,4-D metabolism and resistance to HgCl2. Hybridization of these two plasmids was demonstrated by using nick-translated 32P-labeled pJMP397. The vector pBR325 was used to clone HindIII fragments from pEML159. One cloned fragment of 14.8 megaldaltons expressed in Escherichia coli the ability to release 14CO2 from 2,4-D labeled in the acetate portion.     

Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis

A novel phytase gene ( phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene ( 168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a phi105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 degrees C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.