Species-specific serine-threonine protein kinase Pkb2 of Bifidobacterium longum subsp. longum: Genetic environment and substrate specificity.

The objective of this study was to determine for phosphorylated substrates of the species-specific serine-threonine protein kinase (STPK) Pkb2 from Bifidobacterium longum subsp. longum GT15. Two approaches were employed: analyses of phosphorylated membrane vesicles protein spectra following kinase reactions and analyses of the genes surrounding pkb2. A bioinformatics analysis of the genes surrounding pkb2 found a species-specific gene cluster PFNA in the genomes of 34 different bifidobacterial species. The identified cluster consisted of 5-8 genes depending on the species. The first five genes are characteristic for all considered species. These are the following genes encoding serine-threonine protein kinase (pkb2), fibronectin type III domain-containing protein (fn3), AAA-ATPase (aaa-atp), hypothetical protein with DUF58 domain (duf58) and transglutaminase (tgm). The sixth (protein phosphatase, prpC), seventh (hypothetical protein, BLGT_RS02790), and eighth (FHA domain-containing protein, fha) genes are included in this cluster, but they are not found in all species. The operon organization of the PFNA gene cluster was confirmed with transcriptional analysis. AAA-ATPase, which is encoded by a gene of the PFNA gene cluster, was found to be a substrate of the STPK Pkb2. Fourteen AAA-ATPase sites (seven serine, six threonine, and one tyrosine) phosphorylated by STPK Pkb2 were revealed. Analysis of the spectra of phosphorylated membrane vesicles proteins allowed us to identify eleven proteins that were considered as possible Pkb2 substrates. They belong to several functional classes: proteins involved in transcription and translation; proteins of the F1-domain of the FoF1-ATPase; ABC-transporters; molecular chaperone GroEL; and glutamine synthase, GlnA1. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GlnA1 and FoF1-ATPase alpha and beta subunits) were selected for further in vitro phosphorylation assays and were shown to be phosphorylated by Pkb2. Four phosphorylated substrates of the species-specific STPK Pkb2 from B. longum subsp. longum GT15 were identified for the first time. They included the moonlighting protein glutamine synthase GlnA, FoF1-ATPase alpha and beta subunits, and the chaperone MoxR family of AAA-ATPase. The ability of bifidobacterial STPK to phosphorylate the substrate on serine, threonine, and tyrosine residues was shown for the first time.

Isolation and Purification of Recombinant Serine/Threonine Protein Kinases of the Strain Bifidobacterium longum B379M and Investigation of Their Activity.

Previously, we identified six serine/threonine protein kinases (STPK) of Bifidobacterium and named them Pkb1-Pkb6. In the present study, we optimized methods for isolation of the six STPK catalytic domains proteins of B. longum B379M: a method for isolation of Pkb3 and Pkb4 in native conditions, a method for isolation of Pkb5 in denaturing conditions, and a method for isolation of Pkb1, Pkb2, and Pkb6 from inclusion bodies. The dialysis conditions for the renaturation of the proteins were optimized. All of the enzymes were isolated in quantities sufficient for study of the protein activity. The proteins were homogeneous according to SDS-PAGE. The autophosphorylation ability of Pkb1, Pkb3, Pkb4, and Pkb6 was investigated for the first time. Autophosphorylation was detected only for the Pkb3 catalytic domain.

[The characteristics of probiotic properties of strains of genus of bifidobacterium separated from gastrointestinal tract of residents of the central region of Russia].

The 156 samples of feces separated from healthy residents of the Central region of Russia were used to compose collection of 87 strains of Bifidobacterium out of which 5 strains with wide antagonistic activity related to pathogenic and opportunistic microorganisms were selected. The selected strains are characterized by high probiotic potentials. They have adhesive properties and sensitivity to antibiotics and preparations corresponding to main requirements of common pharmacopoeia articles to strains of microorganisms used in production of probiotics for medicinal application. The given strains of Bifidobacterium can be recommended for development of effective probiotic pharmaceuticals directed to residents of the Central region of Russia.

[Genetic instability of probiotic characteristics in the Bifidobacterium longum subsp. longum B379M strain during cultivation and maintenance].

The stability of inheriting several genes in the Russian commercial strain Bifidobacterium longum subsp. longum B379M during cultivation and maintenance under laboratory conditions has been studied. The examined genes code for probiotic characteristics, such as utilization of several sugars (lacA2 gene, encoding beta-galactosidase; ara gene, encoding arabinosidase; and galA gene, encoding arabinogalactan endo-beta-galactosidase); synthesis of bacteriocins (lans gene, encoding lanthionine synthetase); and mobile gene tet(W), conferring resistance to the antibiotic tetracycline. The other gene families studied include the genes responsible for signal transduction and adaptation to stress conditions in the majority of bacteria (serine/threonine protein kinases and the toxin-antitoxin systems of MazEF and RelBE types) and transcription regulators (genes encoding WhiB family proteins). Genomic DNA was analyzed by PCR using specially selected primers. A loss of the genes galA and tet(W) has been shown. It is proposed to expand the requirements on probiotic strains, namely, to control retention of the key probiotic genes using molecular biological methods.

[Characterization of the transfer-related tra region of the conjugative plasmid p19 from a Bacillus subtilis soil strain].

The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.

[Molecular analysis of some genes from plasmid p19 of the soil strain Bacillus subtilis 19 involved in conjugation].

Two fragments of conjugative plasmid p19 (95 kb) from the soil strain Bacillus subtilis 19 were cloned and sequenced; these fragments carry genes, products of which are indispensable for the conjugative transfer. One of the fragments 4518 bp in size carries five open reading frames and their fragments (ORF1-ORF5). The protein corresponding to ORF4 is homologous to proteins from the family VirD4. Inactivation of ORF4 and ORF1 by insertional mutagenesis caused a three-to-fivefold decrease in the frequency of plasmid p19 conjugative transfer. Another 2932-bp fragment of p19 was shown to possess a rep region homologous to the rep region of plasmid pBS72 from the soil strain B. subtilis 72 and a novel ORF (ORF6); the protein corresponding to this ORF contains the HTH motif typical for DNA-binding proteins.

[Conjugative chromosomal gene transfer in Bacillus subtilis].

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet(r)) and lincomycin (lin(r)) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative pl9cat plasmid producing a hybrid plasmid. Insertion frequency was 10(-4)-10(-5). Then genes tet(r) and lin(r) were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F'- and R' plasmids during conjugation in Escherichia coli and other gram negative bacteria.

[Comparative characterization of the conjugation capacity and large plasmids in native strains of Bacillus subtilis from different regions of the east European plain].

The properties of large plasmids harbored by Bacillus subtilis strains isolated from soils of Moscow and Moscow oblast and from different regions of the Republic of Belarus have been studied. All large plasmids in the collection of strains from Belarus were capable of conjugative mobilization of the small plasmid pUB110 and were similar in size and other properties. Most of the tested plasmids harbored by strains isolated from Moscow soils had no mobilization ability; they were of different sizes and showed no homology with the replication region of plasmids from the Belarussian collection. The uniformity of the plasmids present in strains from Belarussian soils may be due to their active horizontal transfer under natural conditions.

[Inter- and intraspecies conjugal transfer of different plasmids in bacilli]. / Mezhvidovoi i vnutrividovoi kon''iugativnyi perenos razlichnykh plazmid u batsill.

Conjugal transfer of plasmid pUB110 between different strains of bacilli was studied. The plasmid transfer was possible not only between various strains of B. subtilis, but also when many other species of bacilli served as recipients. Conjugation of a donor strain B. subtilis 19 (p19pUB110) was accompanied by a transfer of plasmid p19 along with plasmid pUB110 to the B. subtilis recipient strains lacking a large plasmid p19. If, like the donor cells, the recipient B. subtilis strain carried plasmid p19, the frequency of conjugation decreased. The small plasmid pBC16 was also capable of conjugative transfer. However, if this plasmid carried the mob gene with an inverted region, the frequency of its transmission dramatically decreased. If the donor strain contained another small plasmid, pV, which also carried the mob gene, the efficiency of transmission was partially restored.

[High frequency conjugative mobilization in natural strains of Bacillus subtilis, bearing a large plasmid]. / Kon''iugativnaia mobilizatsiia, osushchestvliaemaia s vysokoi prirodnym shtammom Bacillus subtilis, nesushchim krupnuiu plazmidu.

Conjugative properties of the strain Bacillus subtilis that carrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis 168 strain with a frequency of approximately 10(-2). The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugal mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37 degrees C, but conjugation also proceeded at lower temperatures, up to 21 degrees C.

[A large conjugative plasmid from a soil strain of Bacillus subtilis]. / Krupnaia kon''iugativnaia plazmida iz pochvennogo shtamma Bacillus subtilis.

A large plasmid 35.5 kb in size was found in the soil Bacillus subtilis strain. This plasmids was shown to be capable of conjugal mobilization of small plasmids.

[Simultaneous presence of three cryptic plasmids of various size in a soil strain of Bacillus subtilis]. / Odnovremennoe prisutstvie trekh kripicheskikh plazmid raznoi velichiny v pochvennom shtamme Bacillus subtilis.

The Bacillus subtilis 1387 soil strain, which contains three cryptic plasmids simultaneously, was described. Two small plasmids (6.3 and 8.5 kb) were homologous to each other, and a large plasmid (30 kb) had no homology with them. The plasmids were separately transmitted into cells of the Bac. subtilis 168 strain, and some plasmid characteristics were analyzed.

[RecA-independent induction of the new plasmid rAS1 after introduction of a fragment of the cryptic plasmid p1414 into Bacillus subtilis 168 cells]. / RecA-nezavisimaia induktsiia novoi plazmidy pAS1 posle vvedeniia v kletki Bacillus subtilis 168 fragmentov kripticheskoi plazmidy p1414.

Bacillus subtilis 168 was transformed with fragments from the minireplicon of the cryptic plasmid p1414; these fragments were ligated into the cat gene of plasmid pC194. As a result, the 4.6 kb plasmid pAS1 appeared with a low frequency. The plasmid was homologous to the chromosomal DNA of B. subtilis 168, but had no homology with p1414. Plasmid pAS1 had extensive homology (3.2 kb) with plasmid pUB110 and its restriction map in this region of homology correlated well with the restriction map of pUB110. Plasmid pAS1 occurred both in rec+ and recA- cells. We suppose that pAS1 was generated because of the illegitimate recombination between p1414 and the replicon pUB110 incorporated in the chromosome of B. subtilis 168, and the resulting substitution of marker KmR of pUB110 for marker CmR of pC194.

[Features of transformation of competent cells and Bacillus subtilis protoplasts by integrative vectors]. / Izuchenie osobennostei transformatsii integrativnym vektorami kompetetnykh kletok i protoplastov Bacillus subtilis.

The effect of structural peculiarities of DNAs from integrative plasmids on the transformation activity was studied. Monomeric forms of the plasmids can only transform B. subtilis competent cells, when plasmid selective marker is inserted into chromosomal fragment within the plasmid. Polymeric forms are needed for efficient transformation. Both single- and double-stranded DNAs of integrative plasmids transform no B.subtilis protoplasts, this being irrespective of plasmid structure.

[Integration and expression of the human dihydrofolate reductase gene in the Bacillus subtilis chromosome]. / Integratsiia i ékspressiia gena digidrofolatreduktazy cheloveka v khromosome Bacillus subtilis.

Integration of functionally active human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis chromosome was performed. The clones obtained contained 1 to 7 copies of hDHFR gene per chromosome equivalent and were resistant to trimethoprim. In cell lysates of such clones a protein with the molecular mass of hDHFR was detected. The hDHFR gene was stably maintained in all clones having this gene integrated into the bacterial chromosome, when grown under non-selective conditions.

[Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors]. / Konstruirovanie plazmid, vstraivaiushchikhsia v khromosomu Bacillus subtilis za schet gomologichnoi rekombinatsii, i ispol'zovanie ikh kak integrativnykh vektorov.

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.

Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome.

Integration of expressible DNA corresponding to the human dihydrofolate reductase (hDHFR) gene into the Bacillus subtilis genome has been achieved in different ways. The clones obtained contained one to seven copies of this gene per genome equivalent and were resistant to trimethoprim. Clones produce a new protein coded by the integrated hDHFR gene. In all clones, the integrated DNA was stably maintained even under nonselective growth conditions.