Effect of the Integration of Vector pJFF350 into Plasmid 85-MDa of Azospirillum brasilense Sp245 on Bacterial Flagellation and Motility

Results of genetic analysis of three derivatives of Azospirillum brasilense Sp245 (strains BK570, SK051, and SK248) carrying cointegrates of plasmids 85-MDa and pJFF350 (the vector for omegon mutagenesis), which manifest abnormalities in flagellation and motility, are presented. It was shown for the first time that the integration of the suicide vector into one of Azospirillum resident plasmids is accompanied by the formation of various fusion products and changes in flagellation and motility of these bacteria, such as the loss of the polar (Fla) and lateral (Laf) flagella in SK051; inactivation of Fla and Laf in SK248; and Fla-dependent acceleration of expansion in semiliquid media in BK570.     

Alterations in the primary structure of an 85-MDa plasmid affecting flagellation and motility of bacterium Azospirillum brasilense Sp245

Earlier such Azospirillum brasilense Sp245 mutants as flagellation-defective SK051, SK248 with immobilized flagella, and BK570 swimming and swarming faster than Sp245 were obtained. In SK051 and SK248 the self-killer vector pJFF350 integrated into the 18.3-kb XhoI fragment ofplasmid 85MDa (p85) while in BK570, it integrated into the 9.1-kb XhoI-fragment of p85. In the present work, analysis of the nucleotide sequence of fusion products of p85 and pJFF350 was performed. In p85, in addition to three IS elements (two of which caused cointegrate formation) and phage integrase gene, 22 open reading frames with coding sequence properties were identified. Possible participation of predicted translation products of several p85 genes in bacterial motility detection is discussed. Since differences in the primary structure of p85::pJFF350 cointegrates from SK051 and SK248 cells are localized within pJFF350 DNA, different effects of DNA-folding changes on expression of corresponding p85 genes are suggested.     

Alterations in the primary structure of an 85-MDa plasmid affecting flagellation and motility in the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245

Earlier, such Azospirillum brasilense Sp245 mutants as flagellation-defective SK051, SK248 with paralyzed flagella, and BK570 swimming and swarming faster than Sp245 were obtained. In SK051 and SK248, the suicide vector pJFF350 integrated into the 18.3-kb XhoI fragment of an 85-MDa plasmid (p85) while in BK570, it integrated into the 9.1-kb XhoI-fragment of p85. In the present work, analysis of the nucleotide sequence of fusion products of p85 and pJFF350 was performed. In p85, in addition to three IS elements (two of which mediated cointegrate formation) and phage integrase gene, 22 open reading frames with coding properties were identified. Possible participation of predicted translation products of several p85 genes in determination of bacterial motility is discussed. Since differences in the primary structure of p85::pJFF350 cointegrates from SK051 and SK248 cells are localized within pJFF350 DNA, different effects of DNA-folding changes on the expression of corresponding p85 genes are suggested.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Changes in cell surface properties and biofilm formation efficiency in <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 mutants in the putative genes of lipid metabolism <Emphasis Type="Italic">mmsB1</Emphasis> and <Emphasis Type="Italic">fabG1</Emphasis>

The previously obtained insertion mutants of Azospirillum brasilense Sp245 in the genes mmsB1 and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense strains Sp245, SK039, and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide preparations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.     

Formation of polar bundles of pili on a cell and the behavior of Azospirillum brasilense in semiliquid agar]

This paper describes the formation of single polar bundles of pili on Azospirillum brasilense cells, the twitching motility of cell aggregates, and a new type of social behavior--the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri+ phenotype)--which is an alternative to swarming (the Swa+ phenotype). The wild-type A. brasilense cells occurring in a semiliquid agar may show either the Swa+Gri-, or Swa-Gri-, or Swa-Gri+ phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilense cells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Conjugal Transfer of Plasmid-Encoded Determinants for Bacteriocin Production and Immunity in Lactobacillus acidophilus 88

Lactobacillus acidophilus 88 produced a bacteriocin, designated lactacin F, that demonstrated inhibitory activity toward L. acidophilus 6032, L. lactis 970, L. helveticus 87, L. bulgaricus 1489, L. leichmanii 4797, L. fermentum 1750, and Streptococcus faecalis 19433. Production of lactacin F was pH dependent and could be maximized in MRS broth cultures maintained at pH 7.0. Lactacin F was heat stable and sensitive to ficin, proteinase K, trypsin, and Bacillus subtilis protease. L. acidophilus 88 harbored plasmids of 4 and 27 megadaltons. Variants of L. acidophilus 88 which were deficient in lactacin F production (Laf) and lactacin F immunity (Laf) retained the two resident plasmids. A Laf Laf derivative, L. acidophilus 89, was used as a recipient in agar surface mating experiments with L. acidophilus 88 (Laf Laf). Two types of Laf Laf transconjugants were recovered. One type (T-E) had acquired two plasmids of 68 (pPM68) and 52 (pPM52) megadaltons that were not detected in either the conjugal donor or the other type of Laf Laf transconjugants (T-89). Laf and Laf were unstable in the plasmid-bearing transconjugant. Plasmid analysis of Laf Laf variants revealed that pPM52 and pPM68 were cured with loss of Laf and Laf. Bacteriocin production and immunity phenotypes were genetically stable in Laf Laf transconjugants not harboring pPM52 and pPM68, suggesting chromosomal integration of the transferred determinants. The data demonstrated intragenic conjugation in L. acidophilus and provided direct evidence for involvement of transient plasmid determinants in Laf and Laf.     

Transposon tnPhoA mutagenesis as a method of obtaining mutants of Azospirillum brasilense by motility and flagellation

Three A. brasilense strains (S27, SpBr14, and KR77) did not hydrolyze the chromogenic substrate of alkaline phosphatase (PhoA), X-phosphate, in situ, and were used as recipients in experiments on TnphoA mutagenesis. KMR transconjugates were obtained only for A. brasilense S27, 85% of them were also PhoA+. About 12% TnphoA mutants of A. brasilense S27 had reduces capacity to swarming and 3% of mutants neither swam nor swarmed. These totally immotile clones were examined under transmission electron microscope and were classified as Fla-Laf-, Fla-leakyLaf-, and Fla-Laf+ mutants. In Fla-Laf+ TnphoA mutants of S27, the expression of their lateral flagella (Laf) retained the wild-type inducibility. The presence of intact polar flagellum (Fla) did not seem to be obligatory for controllable expression of Laf in A. brasilense S27. The data suggest that A. brasilense S27 Fla and Laf systems have common structural and/or regulatory components. The PhoA+ phenotype of S27 Fla- mutants suggested a periplasmic and/or membrane localization of the hybrid proteins, the formation of which blocks the flagellar assembly or functioning. Immunochemical analysis with antibodies to alkaline phosphatase will identify these proteins.     

The use of fragments of the 85- and 120-MDa plasmids of Azospirillum brasilense Sp245 to study the plasmid rearrangement in this bacterium and to search for homologous sequences in plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of the Azospirillum brasilense Sp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilense Sp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

The Use of Fragments of the 85- and 120-MDa Plasmids of Azospirillum brasilense Sp245 to Study the Plasmid Rearrangement in This Bacterium and to Search for Homologous Sequences in Plasmids of Azospirillum brasilense Sp7

A spontaneous loss of the 85- (p85) and 120-MDa (p120) replicons and simultaneous generation of a plasmid of more than 300 MDa were associated with defects in synthesis of O-specific and Calcofluor-binding polysaccharides and had no effect on flagellation and motility of theAzospirillum brasilenseSp245.5 mutant. The plasmid rearrangement was studied by hybridization of DNAs from the wild-type Sp245 strain and the Sp245.5 mutant with p85 and p120 fragments that contained loci involved in formation of the polar (fla) and lateral (laf) flagella, synthesis of O-specific and Calcofluor-binding polysaccharides (lps/cal), swimming (mot), and swarming (swa) of bacteria. Hybridization with the p120 fragments revealed incorporation of the intact fla/swa loci and the altered lps/cal loci into a new megaplasmid. Two EcoRI fragments homologous to the fla/laf/mot/swa loci of p85 were found in A. brasilense Sp245 DNA, whereas only one copy was preserved in the Sp245.5 mutant. Hybridization of the p120 and p85 fragments of Sp245 to the A. brasilenseSp7 DNA for the first time revealed regions of substantial homology to these fragments in the 90- and 115-MDa Sp7 plasmids, respectively.     

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM’s effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM’s effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM’s effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.     

Structure-function relationship of bifunctional scorpion toxin BmBKTx1

As the first identified scorpion toxin active on both big conductance Ca2+-activated K+ channels (BK) and small conductance Ca2+-activated K+ channels (SK), BmBKTx1 has been proposed to have two separate functional faces for two targets. To investigate this hypothesis, two double mutants, K21A-Y30A and R9A-K11A, together with wild-type toxin were expressed in Escherichia coli. The recombinant toxins were tested on cockroach BK and rat SK2 channel for functional assay. Mutant K21A-Y30A had a dramatic loss of function on BK but retained its function on SK. Mutant R9A-K11A did not lose function on BK or SK. These data support the two functional-face hypothesis and indicate that the BK face is on the C-terminal beta-sheet.     

Negative feedback regulation of nerve-mediated contractions by KCa channels in mouse urinary bladder smooth muscle

When the urinary bladder is full, activation of parasympathetic nerves causes release of neurotransmitters that induce forceful contraction of the detrusor muscle, leading to urine voiding. The roles of ion channels that regulate contractility of urinary bladder smooth muscle (UBSM) in response to activation of parasympathetic nerves are not well known. The present study was designed to characterize the role of large (BK)- and small-conductance (SK) Ca(2+)-activated K(+) (K(Ca)) channels in regulating UBSM contractility in response to physiological levels of nerve stimulation in UBSM strips from mice. Nerve-evoked contractions were induced by electric field stimulation (0.5-50 Hz) in isolated strips of UBSM. BK and SK channel inhibition substantially increased the amplitude of nerve-evoked contractions up to 2.45 +/- 0.12- and 2.99 +/- 0.25-fold, respectively. When both SK and BK channels were inhibited, the combined response was additive. Inhibition of L-type voltage-dependent Ca(2+) channels (VDCCs) in UBSM inhibited nerve-evoked contractions by 92.3 +/- 2.0%. These results suggest that SK and BK channels are part of two distinct negative feedback pathways that limit UBSM contractility in response to nerve stimulation by modulating the activity of VDCCs. Dysfunctional regulation of UBSM contractility by alterations in BK/SK channel expression or function may underlie pathologies such as overactive bladder.     

Effects of Related Anionic Detergents on Flagellation, Motility, Swarming, and Growth of Proteus

The effects of a series of sodium alkyl sulfates (C(4) to C(16)) on flagellation, motility, swarming, and growth of Proteus were examined. The concentrations of the various sodium alkyl sulfates completely inhibiting the swarming phenomenon (on solid medium) and motility (in liquid medium) were in the same order of magnitude. The inhibiting effect of the detergents examined increased from sodium hexyl sulfate (inhibitory concentration, 20 to 30 mmoles per liter) to sodium tetradecyl sulfate (inhibitory concentration, 0.1 to 0.5 mmoles per liter). Flagella were produced neither in liquid nor on solid medium at these concentrations as could be observed by electron microscopy. At concentrations where motility was not impaired, intact flagellation could be observed. At a concentration of 0.1 mmole per liter, sodium tetradecyl sulfate completely inhibited the motility of Proteus in the liquid medium employed without impairing growth.     

Spontaneous Super-Swarming Derivatives of Azospirillum brasilense Sp245 have Different DNA Profiles and Behavior in the Presence of Various Nitrogen Sources

Azospirillum brasilense swims in liquid environments and swarms in semisolid media. Five variants of A. brasilense Sp245, Sp245.P1–Sp245.P5, which swarmed faster than Sp245 in a semisolid malate–salt medium, have been isolated. In Sp245.P1–Sp245.P4, a new megaplasmid was revealed instead of an indigenous 85-MDa plasmid (p85). By polymerase chain reactions (PCR) with primers to the segments of p85 important for proper bacterial motility/flagellation and for dissimilatory nitrite and NO reduction, that DNA of p85 was found retained by all the variants. In ERIC- and RAPD-PCR, microdiversity between the total DNAs of Sp245 and its variants was detected. Interstrain differences in growth characteristics in liquid peptone–succinate–salt medium with KNO₃ or KNO₂ and in KNO₂ production/consumption were revealed. Although all the variants swam and swarmed faster than Sp245 in the medium supplemented with NH₄Cl or KNO₃, not all of them could do so in MPSS with KNO₂.     

Plasmid visualization and nif gene location in nitrogen-fixing Azospirillum strains.

A modified gel electrophoresis technique provided a reproducible way of detecting and isolating plasmids with molecular weights ranging from 12 X 10(6) to 370 X 10(6) for Azospirillum species. Analysis with the nifHD region of Rhizobium trifolii showed that the Azospirillum nif genes were chromosomally located in all eight strains investigated and not on endogenous plasmids.     

Functional Upregulation of Ca2+ -Activated K+ Channels in the Development of Substantia Nigra Dopamine Neurons

Many connections in the basal ganglia are made around birth when animals are exposed to a host of new affective, cognitive, and sensori-motor stimuli. It is thought that dopamine modulates cortico-striatal synapses that result in the strengthening of those connections that lead to desired outcomes. We propose that there must be a time before which stimuli cannot be processed into functional connections, otherwise it would imply an effective link between stimulus, response, and reward in uterus. Consistent with these ideas, we present evidence that early in development dopamine neurons are electrically immature and do not produce high-frequency firing in response to salient stimuli. We ask first, what makes dopamine neurons immature? and second, what are the implications of this immaturity for the basal ganglia? As an answer to the first question, we find that at birth the outward current is small (3nS-V), insensitive to , TEA, BK, and SK blockers. Rapidly after birth, the outward current increases to 15nS-V and becomes sensitive to , TEA, BK, and SK blockers. We make a detailed analysis of the kinetics of the components of the outward currents and produce a model for BK and SK channels that we use to reproduce the outward current, and to infer the geometrical arrangement of BK and channels in clusters. In the first cluster, T-type and BK channels are coupled within distances of 20 nm (200 Å). The second cluster consists of L-type and BK channels that are spread over distances of at least 60 nm. As for the second question, we propose that early in development, the mechanism of action selection is in a “locked-in” state that would prevent dopamine neurons from reinforcing cortico-striatal synapses that do not have a functional experiential-based value.