[Morphological changes in brain tissues of rat with local permanent ischemia model under the action of 2-methyl-6-ethyl-3-hydroxypyridine hemisuccinate].

The influence of 2-methyl-6-ethyl-3-hydroxypiridine hemisuccinate on the morphological state of rat brain tissue after the occlusion of left middle cerebral artery has been studied. It was found that 6- and 12-day treatment with 2-methyl-6-ethyl-3-hydroxypyridine hemisuccinate at a dose of 100 mg/kg (intraperitoneal) led to regenerative processes in ischemic brain tissue. The latter treatment duration was most effective.

Bacterial community structure in tree hole habitats of Ochlerotatus triseriatus: influences of larval feeding.

We investigated the bacterial community composition of tree holes in relation to the presence and absence of larvae of the mosquito Ochlerotatus triseriatus. Larvae were eliminated from a subset of natural tree holes with Bacillus thuringiensis serovar israelensis, and total bacterial numbers, slow- and fast-growing colony-forming units on minimal media, and 16S rRNA gene sequence data from water column and leaf material were obtained. Total bacterial counts did not change significantly with treatment; however, the number of slow-growing cultivable bacteria significantly increased in the absence of larvae. Sequence classifications and comparisons of sequence libraries using LIBSHUFF indicated that the elimination of larvae significantly altered bacterial community composition. Major groups apparently affected by larvae were Flavobacteriaceae, Rhodobacteraceae, Comamonadaceae, and Sphingomonadaceae. A clear dominance of Flavobacteriaceae in the water column after larval removal suggests members of this group are a major bacterial food source.

Horizontal transfer of Shiga toxin and antibiotic resistance genes among Escherichia coli strains in house fly (Diptera: Muscidae) gut.

Whether the house fly, Musca domestica L., gut is a permissive environment for horizontal transfer of antibiotic resistance and virulence genes between strains of Escherichia coli is not known. House flies were immobilized and force fed suspensions of defined, donor strains of E. coli containing chloramphenicol resistance genes on a plasmid, or lysogenic, bacteriophage-born Shiga toxin gene stx1 (bacteriophage H-19B::Ap1). Recipient strains were E. coli lacking these mobile elements and genes but having rifampicin as a selectable marker. Plasmid transfer occurred at rates of 10(-2) per donor cell in the fly midgut and 10(-3) in the fly crop after 1 h of incubation postfeeding. Bacteriophage transfer rate was approximately 10(-6) per donor cell without induction, but induction with mitomycin C increased rates of transfer to 10(-2) per donor cell. These findings show that genes encoding antibiotic resistance or toxins will transfer horizontally among bacteria in the house fly gut via plasmid transfer or phage transduction. The house fly gut may provide a favorable environment for the evolution and emergence of pathogenic bacterial strains through acquisition of antibiotic resistance genes or virulence factors.

Secretion of recombinant proteins by Gram-negative bacteria.

During the past few years, significant progress has been made towards our understanding of the molecular mechanisms governing the translocation of proteins through bacterial cell membranes. Successful attempts in promoting the secretion of recombinant proteins by employing this knowledge and by empirical efforts have been registered. However, a further in-depth understanding of membrane-translocation mechanisms is required before predictable manipulations of secretion systems can be made to secrete native recombinant proteins that are not naturally targeted to the extracellular compartment.

Replication and copy number control of the broad-host-range plasmid RSF1010.

Initiation of replication of the broad-host-range plasmid RSF1010, is accurately controlled by the plasmid-encoded proteins, RepB (MobA), RepB', RepA and RepC [Haring et al., Proc. Natl. Acad. Sci. USA 82 (1985) 6090-6094; Scherzinger et al., Nucleic Acids Res. 19 (1991) 1203-1211]. The genes encoding these proteins which are essential for replication and conjugative mobilization are transcribed from a cluster of promoters, P1/P3 and P2, which partly overlap with the origin of conjugal transfer, oriT. Three regions were found where deletion mutations affect the mobilization of RSF1010 and increase its copy number in Escherichia coli. A deletion in the mobC gene increased the copy number of RSF1010 four-fold. Another deletion, that removed oriT and part of the promoter believed to be responsible for the expression of mobC, results in a three-fold increase in copy number. The third type of deletions affect the N-terminal part of RepB (MobA). A deletion that created a frame-shift results in a three-fold increase in copy number. A smaller, in-frame deletion of this region only affected the mobilization of RSF1010, but not its copy number. The extent by which RSF1010 or its deletion derivatives could repress the P1/P3 and P2 promoters has indicated that these promoters are negatively regulated by MobC and RepB (MobA), presumably by their attachment to the oriT region of RSF1010. Both MobC and RepB are required for the maximal repression of the rep operon. Optimal function of RSF1010 thus involves not only overlapping genes, but also proteins that exert multiple functions, mobilization, replication and regulation.

Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors.

A broad-host-range vector, pKT240, containing the structural gene (aph) for aminoglycoside phosphotransferase (APH), without promoter, has been constructed. Insertion of DNA fragments carrying promoters upstream of aph gene into the unique EcoRI site of this vector results in the expression of the aph gene and consequently the resistance of the host cells to streptomycin. The new vector has been used to show that the hybrid trp-lac (tac) promoter and the promoter of the lacIQ gene of Escherichia coli are active in Pseudomonas putida. Derivatives of pKT240 containing tac and lacIQ sequences may be used as wide-host-range expression vectors. Regulated overproduction of APH and catechol 2,3-oxygenase can be obtained with the aid of the new vectors in both E. coli and P. putida.

A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae.

A gene essential for the secretion of cholera toxin from the periplasm of Vibrio cholerae into the extracellular medium has been isolated and its nucleotide sequence determined. It encodes a cytoplasmic protein of 56 kDa that exhibits a high degree of similarity to gene products required for extracellular protein secretion in several other Gram- organisms. Sequence similarities in its potential ATP-binding site suggest that the protein may act as an energy provider or signal transducer in the process of extracellular secretion.

RepB' is required in trans for the two single-strand DNA initiation signals in oriV of plasmid RSF1010.

We have shown previously [Honda et al., Gene 68 (1988) 221-228] that the oriV region of the broad-host-range plasmid RSF1010 contained two single-strand DNA initiation signals (ssi) which have RSF1010-specific properties since they required one or more factors provided in trans by RSF1010 for their functional activities. We demonstrate here, by deletion analysis, that repB', one of the genes essential for the vegetative replication of RSF1010, produces a factor required for the function of ssi signals. It is conceivable that the RepB' protein and the two ssi signals, ssiA and ssiB, cooperatively compose plasmid-specific priming complexes which confer the broad-host-range property upon RSF1010.

A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants.

A series of controlled expression vectors was constructed based on the wide-host-range plasmid pMMB66EH. Some of these new vectors code for the alpha-peptide of beta-galactosidase and allow the direct screening of recombinant clones by inactivation of alpha-complementation. The bla gene was replaced in some plasmids by the cat gene of Tn9 coding for chloramphenicol resistance, extending the use into beta-lactam-resistant strains. They all feature either the tac or taclac (tac-lac UV5 in tandem) promoters in front of a polylinker followed by the rrnB transcriptional stop point. These vectors were tested by subcloning the xylE gene coding for the Pseudomonas putida catechol 2,3-oxygenase and the Escherichia coli lamB gene coding for the lambda receptor. The expression of these genes in E. coli indicated that the tac promoter is five times stronger than the taclac promoter and that both were tightly regulated. The tac promoter in Pseudomonas syringae pv glycinea and Xanthomonas campestris pv vesicatoria had a strength similar to that in E. coli, while the taclac promoter was much weaker, reaching only 6.5 and 3% of the level of expression of the tac promoter, respectively. The taclac promoter, however, proved to be useful for the cloning in E. coli of DNA fragments that were unstable in vectors with stronger promoters and higher copy number. Expression of the lamB gene in Vibrio cholerae strain TRH7000 was not sufficient to permit cosmid transduction. Two subunits of the E. coli mannose permease, coded by the ptsP and ptsM genes, are also required for cosmid DNA penetration into the recipient cells.

Specificity of the protein secretory apparatus: secretion of the heat-labile enterotoxin B subunit pentamers by different species of gram- bacteria.

The B-subunit pentamer(s) (EtxBp) of Escherichia coli heat-labile enterotoxin (LT) are secreted from Vibrio cholerae via the general secretion pathway (GSP), but remain periplasmic in E. coli. In order to determine if other Gram- bacteria were also able to secrete the ExtBp, the etxB gene, which encodes EtxB was introduced into different bacteria. Of the bacteria examined, most species of Vibrio and Aeromonas were able to secrete this protein through the outer membrane; other Gram- genera, including Erwinia, Klebsiella and Xanthomonas were not, even though they encode GSP genes homologous to those of V. cholerae. Thus, the ability to recognize the EtxBp as a secretable protein is confined to bacteria that were identified as being closely related to V. cholerae by examination of their 5S rRNA [MacDonell and Colwell, Syst. Appl. Microbiol. 6 (1985) 171-182].

Genes required for extracellular secretion of enterotoxin are clustered in Vibrio cholerae.

Pleiotropic transposon insertion mutants of Vibrio cholerae that are unable to secrete enterotoxin, HA/protease and chitinase through the outer membrane have been isolated. The gene, epsM, responsible for complementation of two of the Tn5 insertion mutations was sequenced. It encodes a putative cytoplasmic membrane protein of 18.5 kDa that exhibits similarity to proteins required for extracellular secretion of pullulanase, pectate lyase or elastase in other Gram-bacteria. It is present on a 15-kb DNA fragment from the V. cholerae genome, containing the epsE gene that was previously shown to be required for secretion of cholera toxin [Sandkvist et al., Gene 123 (1993) 81-86]. Partial reading frames flanking epsM also demonstrated similarity to genes required for extracellular secretion of pullulanase in Klebsiella oxytoca.

Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria.

A cosmid cloning system has been developed which is useful for the construction of genomic libraries and the introduction of clones into a broad range of bacterial species. The cosmids pMMB33 and pMMB34 allow selective cloning into their unique BamHI site of 36-kb DNA fragments generated by BamHI, Sau3A and MboI partial digestion. This selective cloning is achieved by a strategy that avoids formation of polycosmids without a dephosphorylation step. It uses two unique recognition sites within the vectors for endoncleases that generate blunt-ended DNA fragments for the preparation of left and right cosmid "arms". An alternative method that uses the unique EcoRI and SstI sites and dephosphorylation of the cosmid arms prior to BamHI digestion is also outlined and discussed. The DNA is first cloned with either vector into a rec- E. coli strain, where clones can be maintained stably, and can then be introduced by mobilization into a wide range of Gram-negative species to permit the study of gene expression and complementation. Because mobilization is much more efficient than transformation, the vector has the advantage that it can be transferred between bacterial species that specify different restriction systems, where transformation appears to be inefficient. The vectors have been used to generate gene libraries from the chromosomal DNA of several Pseudomonas and a Thiobacillus species. The genes specifying myo-inositol transport from Pseudomonas strain JD34 have been cloned with this system.

Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.

Host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. They comprise restriction-negative host strains of Pseudomonas aeruginosa and P. putida and new cloning vectors derived from the high-copy-number, broad-host-range plasmid RSF1010, which are stably maintained in a wide range of Gram-negative bacteria. These plasmids contain EcoRI, SstI, HindIII, XmaI, XhoI, SalI, BamHI, and ClaI insertion sites. All cloning sites, except for BamHI and ClaI, are located within antibiotic-resistance genes' insertional inactivation of these genes during hybrid plasmid formation provides a readily scored phenotypic change for the rapid identification of bacterial clones carrying such hybrids. One of the new vector plasmids is a cosmid that may be used for the selective cloning of large DNA fragments by in vitro lambda packaging. An analogous series of vectors that are defective in their plasmid-mobilization function, and that exhibit a degree of biological containment comparable to that of current Escherichia coli vector plasmids, are also described.

Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector.

Plasmid RP4 primase was overproduced by utilizing autoregulated high-level expression vector systems in Escherichia coli and in four other Gram-negative bacterial species. Analysis of the products in E. coli revealed that in addition to the two primase polypeptides of 118 and 80 kDa the pri region of RP4 encodes two smaller proteins of 16.5 and 8.6 kDa. The transcript for the four RP4-specified products is polycistronic. The vector system used in E. coli is based on the plasmid pKK223-3 (Brosius and Holy, 1984), a ColE1-type replicon which contains a polylinker sequence flanked on one side by the controllable tac promoter and on the other side by two strong transcriptional terminators. The gene for the lac repressor (lacIQ) was inserted to render the use of the plasmid independent from repressor-overproducing strains. The gene cartridge essential for high-level expression and selection was combined with the RSF1010 replicon to generate a vector plasmid functioning in a wide variety of Gram-negative hosts. The versatility of the vector family was extended by constructing derivatives that contain the polylinker in inverted orientation relative to the tac promoter. Therefore, the orientation of the cloned fragment can be chosen by 'forced cloning' into the appropriately selected vector.

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010.

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

A dnaA box can functionally substitute for the priming signals in the oriV of the broad host-range plasmid RSF1010.

The initiation of replication from oriV RSF1010, the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB' (primase), and RepC (initiator protein), encoded by RSF1010 itself, while this initiation event in E. coli is independent of dnaA, dnaB, dnaC, and dnaG [Scherzinger et al. (1984) Proc. Natl. Acad. Sci. USA 81, 654-658; Scholz et al. (1985) in: Plasmids in Bacteria, pp. 243-259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp. 95-124, Academic Press, London; Scherzinger et al. (1991) Nucl. Acids Res. 19, 1203-1211]. We showed in this work that a newly constructed origin consisting of an oriV RSF1010 and a DnaA protein binding site, the dnaA box, inserted near oriV RSF1010 (oriV RSF1010-dnaA box) could function without RepB' primase, but required RepA and RepC. This oriV RsF1010-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature. These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB' dependent priming signals in oriV RSF1010, and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.

Integration of F factor and cryptic LT2 plasmid into a specific site of the Salmonella typhimurium chromosome.

Suppression of dnaA mutation by F'lac and cryptic LT2 plasmid was used for selecting clones containing these plasmids integrated into rfa and galK genes.

[The effect of the addition of Co2+ ions on the osmolarity and concentration of Na+, K+ and Ca2+ ions in the blood serum of rats]. / Vliianie dobavleniia ionov Co2+ na osmoliarnost' i kontsentratsiiu ionov Na+, K+, Ca2+ v syvorotke krovi u krys.

The addition of cobalt chloride to the female rat blood serum resulted in a decrease of serum osmolality and in an increase of ionized calcium concentration probably due to ousting of calcium out of proteins and alteration of proteins characteristics. The osmolality of saline and activities of sodium, potassium and calcium in it did not change following addition of Co2+. The administration of vasopressin and dehydration were not followed by alteration of characteristics of serum reaction in response to addition of Co2+ in rats. More obvious effect of Co2+ was revealed with high initial serum osmolality.