Non-induced Bacteria Research Articles

Production, Purification and Characterization of the Recombinant Brucella abortus rP17 Protein

Immunoreactive cytosolic P17 protein of Brucella abortus was produced in Escherichia coli as 6xHistidine tagged recombinant protein (rP17) by cloning the p17 gene into pColdI cold-shock expression vector. DNA sequence analysis of the cloned p17 gene showed that the recombinant rP17 protein contains a total of 181 amino acids constituted of 83 hydrophobic, 42 hydrophilic, 35 basic and 21 acidic residues. Its theoretical isoelectric point was calculated as 6.42 and GRAVY index of -0.097 indicates its solubility. The instability index classifi es the rP17 as a stable protein expressed in the transformed E. coli cells by inducing with IPTG. Lysate of the induced and non-induced bacteria was analyzed by SDS-PAGE showing expression of the rP17 with a relative molecular weight of 24 kDa. After two-step purifi cation procedure, Ni-NTA affi nity chromatography and elution from polyacrylamide gels following SDS-PAGE, the rP17 was highly purifi ed and analyzed by Western blot. Preliminary results showed that the recombinant rP17 protein still preserves its immunoreactivity. In present, large scale production of the rP17 is carried out for evaluation of its diagnostic performance with a large panel of well-defi ned sera.

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and class switch recombination of Ig genes in B cells. Although AID has been shown to deaminate deoxycytidine to deoxyuridine in DNA in vitro, there is no physical evidence for increased uracils in DNA from cells expressing AID in vivo. We used several techniques to detect uracil bases in a gene that was actively transcribed in Escherichia coli cells expressing AID. Plasmid DNA containing the gene was digested with uracil-DNA glycosylase to remove uracil, and apurinic/apryimidinic endonuclease to nick the abasic site. The nicked DNA was first analyzed using alkaline gel electrophoresis, in which there was a 2-fold increase in the linear form of the plasmid after AID induction compared with plasmid from noninduced bacteria. Second, using a quantitative denaturing Southern blot technique, the gene was predominantly nicked in the nontranscribed strand compared with the transcribed strand. Third, using ligation-mediated PCR, the nicks were mapped on the nontranscribed strand and were located primarily at cytosine bases. These data present direct evidence for the presence of uracils in DNA from cells that are induced to express AID, and they are preferentially generated at cytosines in the nontranscribed strand during transcription.

Effects of non-ionic surfactants on the uptake and hydrolysis of fluoresceindiacetate by alkane-oxidizing bacteria

Biological effects of non-ionic surfactants on alkane-oxidizing bacteria were studied by assessing their influence on the uptake of prefluorochrome fluoresceindiacetate (FDA) and its intracellular hydrolysis to fluorescein. Both decreasing and increasing rates of hydrolysis as a consequence of the presence of surfactants were observed. The surfactants influenced the uptake of FDA, but not its intracellular hydrolysis. The effects of the surfactants on the uptake rate depended strongly on the structure and physico-chemical properties of the surfactants. There was no qualitative or significant quantitative difference in surfactant susceptibility between induced (alkane grown) and non-induced bacteria (acetate grown), even though the induced cells possess greater cell surface hydrophobicity.

Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues.

Alternative splicing of hMSH4: two isoforms in testis and abnormal transcripts in somatic tissues.

Establishment of Parameters for Agrobacterium Transformation of Asparagus Embryogenic Suspension Culture Cells

Conditions for Agrobacterium transformation of asparagus embryogenic suspension cells were investigated using an intron-containing GUS gene in pCNL56 to detect transformation events. Embryogenic suspension cultures of Rutgers 22 were maintained on LS medium with 5 mg NAA/liter, subcultured weekly, and used 5 days thereafter. For initial experiments, cells were inoculated at 5 x 108 cfu/ml for 15 min and cocultivated for 4 days on LS medium with 10 g Phytagel/liter (EM1 medium). Subsequently, the cells were transferred to EM1 with 200 mg Timentin/liter and tested for GUS expression after a total of 6 days. The effect of acetosyringone (AS) on four A. tumefaciens strains was tested. With or without AS induction, EHA105 and C58C1(pMP90) produced a significantly greater number of GUS foci on embryogenic cells than C58C1(pGV2260) and LBA4404. Transient expression was very low from cells inoculated with C58C1(pGV2260) and nonexistent from LBA4404. AS induced significantly more GUS foci from EHA105 and C58C1(pMP90) than from the same noninduced bacteria; however, it had no effect on C58C1(pGV2260). Upon repeating the experiment using EHA105 and C58C1(pMP90) only, no differences in response were observed, although AS induced more GUS foci from both strains and EHA105 outperformed C58C1. Inoculum density was investigated using induced EHA105. 5 x 107 cfu/ml significantly increased the number of GUS foci than 108, 5 x 108, or 109, although it was not statistically different from 107, which produced slightly fewer foci.

Cell-surface collagen-binding protein in the procaryote Achromobacter iophagus

Collagen and its high-molecular-weight fragments specifically induce an extracellular collagenase (EC 3.4.24.8) in the Gram-negative Achromobacter iophagus. During the induction process the inducer is concentrated on the bacterial outer membrane. Two-dimensional electrophoresis of 125I-labelled outer membrane proteins has shown that, in particular, the amount of one protein which is already present on the surface of non-induced bacteria increases quantitatively when the inducer is added. After 125I-labelling of the cell membrane and its solubilization, the same protein is retained selectively on a gelatin-Sepharose column. It has isoelectric point of 4.9–5.1 and molecular weight of 40 000. This molecular weight is close to that of the 35 000 of the collagenase subunit. However, their non-identity was proved in three independent ways: upon two-dimensional electrophoresis, only those proteins in the range corresponding to the collagenase dimer ( M r 70 000–80 000) react with fluorescent anticollagenase antibody system, whereas the spot of the collagen-binding protein ( M r 40 000) is negative; the solubilized collagen-binding protein is not retained by anticollagenase-Sepharose affinity chromatography; in vivo, it is not protected by anti-collagenase antibodies against lactoperoxidase iodination. A hypothesis for the possible role of the collagen-binding protein in the induction of collagenase is proposed.

Colicins E4, E5, E6 and A and properties of btuB+ colicinogenic transconjugants.

E colicins are those inactive on btuB mutants, which lack the outer membrane protein that adsorbs vitamin B12, E colicins and phage BF23; types E1, E2, E3 and E7 have been defined by the specific immunity of E-colicinogenic strains to the type of E colicin they produce. Further immunity types - E4, E5, E6 - are now described. Shigella sonnei of colicin type 9 makes colicin E4, and Shigella sonnei of types 9A, 12 and 14 make colicin E6 (not colicin E3, as previously supposed). Many local Shigella sonnei isolates make E colicin of a new type - E5. The action of colicins E1 to E7 was (incompletely) blocked by vitamin B12, which also reduced the effect of A colicins (which are weakly active on Escherichia coli K12 btuB indicators). Escherichia coli K 12 given plasmid Co1A-23 by transformation was immune to A colicins but sensitive to colicins E1 to E7. A purported 'colicin E4' was shown to be of class colicin A. Escherichia coli K 12(CloDF13) transformants were immune to colicin E6. A 'cap' of colicin-sensitive indicator bacteria developed over, but not around, killed colonies of btuB+ E-colicinogenic strains, because of adsorption of colicin by non-induced bacteria of the colony. Killed colonies of btuB+ E-colicinogenic strains gave partly discrete lobes of colicin action on indicator lawns, instead of circular zones, apparently as a result of unstable variation in colicin production. The presence of a colicin E4, E5 or E6 plasmid in K12 btuB+ strains made them more sensitive to colicins E2 and E7, as shown by zones of partial translucency surrounding the ordinary colicin zones and by increased titre of colicin E2 and E7 preparations on such indicators.

Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn.

Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC. The transducing phages had been isolated from a strain of E. coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster [Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1978) Proc. Natl. Acad. Sci. USA 75, 5099--5103]. When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells. In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC. The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC. In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria. These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides. Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes.

The effect of chloramphenicol on the induction of cyanase in Escherichia coli.

The early stages of cyanase induction were studied. It was found that 30 s elapsed at 37 °C and 60 s elapsed at 22 °C before cyanase-forming capacity of Escherichia coli could be demonstrated. This period was termed inducer-dependent stage (i.d.s.). An additional period of 120 s at 22 °C was needed for demonstration of enzyme synthesis. This period was termed inducer-independent stage (i.i.s.). It was found that in contrast to β-galactosidase, the i.d.s. as well as the i.i.s. were inhibited by chloramphenicol. Pulse-labelling experiments showed an increase in m-RNA obtained from induced bacteria in the presence of chloramphenicol compared with noninduced bacteria treated with chloramphenicol, indicating that chloramphenicol does not interfere with transcription. However, the m-RNA formed in the presence of chloramphenicol did not participate in translation upon removal of the antibiotic. This evidence, in addition to the time course of the inhibition, suggests that the inhibition of induction is caused by chloramphenicol preventing the attachment of the ribosomes to cyanase-specific m-RNA.

Colicin K. 8. The immunological properties of mitomycin-induced colicin K.

Partially purified colicin K (mCol K) has been obtained from cultures of Escherichia coli K235 induced with mitomycin C. Unlike colicin K (Col K) derived from noninduced cultures of E. coli K235, mitomycin-induced colicin K (mCol K) is not associated with the type O-specific antigen of the colicinogenic bacillus. mCol K elicits in rabbits specific antibodies which precipitate and neutralize the homologous bacteriocin. These colicin-specific antibodies are not precipitated by the colicin-O antigen complex derived from noninduced bacteria. Colicin-neutralizing antibodies can be separated by zone electrophoresis into fractions having different electrophoretic mobilities. The antibodies with lower mobility strongly precipitate the homologous antigen mCol K; those with higher mobility neutralize the bacteriocin and form soluble antigen-antibody complexes.

The Effect of 8-Azaguanine on the Inducible Oxidation of Guanine by Pseudomonas aeruginosa

SUMMARY: Pseudomonas aeruginosa NCTC 8203 was shown to metabolize guanine by deamination to xanthine, which was then oxidized to uric acid and further products. Guanine deaminase activity was present in non-induced bacteria but was 3-4 times greater in guanine-induced bacteria. Xanthine oxidase, and the uric acid oxidizing enzymes, were not found in bacteria grown in a minimal salts medium but were induced by adding guanine or xanthine to the culture medium towards the end of the growth period. When uric acid was added, only the uric acid oxidizing enzymes were induced. Enzyme induction also occurred when washed suspensions of the bacteria were incubated with guanine, xanthine or uric acid in Warburg flasks. 8-Azaguanine was deaminated more rapidly than guanine by non-induced bacteria and the rate was again greater with guanine-induced bacteria. 8-Azaguanine and 8-azaxanthine were not oxidized and did not induce the synthesis of deaminases or oxidases. 8-Azaguanine and 8-azaxanthine had no effect on the oxidation of guanine, xanthine or uric acid by fully induced bacteria. Equimolar concentrations of 8-azaguanine inhibited adaptation to the oxidation of guanine, xanthine and uric acid when added before the substrates. 8-Azaxanthine, under the same conditions, delayed adaptation to guanine and xanthine oxidation, and completely inhibited adaptation to uric acid. Mutant strains were isolated which were resistant to 10 mM-8-azaguanine and 5 mM-8-azaxanthine which completely inhibited growth of the parent strain. Another class of mutants was isolated resistant to 8-azaguanine and not resistant to 8-azaxanthine. Both classes of mutants were indistinguishable from the parent strain in their susceptibility to 8-azaguanine and 8-azaxanthine inhibition of adaptation to purine oxidation.

INDUCTION AND REPRESSION OF PSEUDOMONAS AERUGINOSA AMIDASE.

The synthesis of an inducible amidase by Pseudomonas aeruginosa 8602/a was studied in cultures growing exponentially in succinate medium. Induction by both the substrate inducer acetamide, and the non-substrate inducer N-acetylacetamide, was repressed by cyanoacetamide. Induction by 10−2mN-acetylacetamide was significantly repressed by 10−4m-cyanoacetamide, but repression of induction by 10−3m-acetamide required a tenfold excess of cyanoacetamide. Amidase synthesis in a medium in which acetamide was the sole carbon + nitrogen source was also repressed by cyanoacetamide, which under these conditions inhibited the growth of non-induced bacteria. Several tricarboxylic acid cycle intermediates, and related compounds, repressed amidase synthesis in exponentially growing organisms. Catabolite repression by propionate in succinate medium was decreased by increasing the concentration of acetamide. These findings are discussed in relation to general theories of regulation of microbial enzyme synthesis.