The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

Modulation of inv gene expression by the OmpR two-component response regulator protein of Yersinia enterocolitica.

To elucidate the physiological meaning of OmpR-dependent expression of invasin gene (inv) inhibition in Yersinia enterocolitica, the function of the EnvZ/OmpR regulatory pathway in osmoregulation of inv expression was analyzed in detail. The osmoregulation of inv expression was found to be a multifaceted process involving both OmpR-dependent and -independent mechanisms. Analysis of inv transcription in strains lacking OmpR or EnvZ proteins indicated that kinase EnvZ is not the only regulator of OmpR phosphorylation. Using the transcriptional inv::lacZ fusion in a heterologous system (Escherichia coli) we tried to clarify the role of OmpR in the inv regulatory circuit composed of negative (H-NS) and positive (RovA) regulators of inv gene transcription. We were able to show a significant increase in inv expression in E. coli ompR background under H-NS( Ecoli )-repressed condition. Moreover, H-NS-mediated inv repression was relieved when RovA of Y. enterocolitica was expressed from a plasmid. Furthermore, we showed that RovA may activate inv expression irrespective on the presence of H-NS protein. Using this strategy we showed that OmpR of Y. enterocolitica decrease RovA-mediated inv activation.

The YompC protein of Yersinia enterocolitica: molecular and physiological characterization.

The structural gene coding for YompC has been identified in the genome of a pathogenic strain of Yersinia enterocolitica O:9, and was subsequently cloned and sequenced. Detailed alignment of the deduced amino acid sequence showed that YompC is a member of the OmpC porin family with the highest degree of homology to Klebsiella pneumoniae. The mutant lacking YompC porin was constructed by insertional inactivation of the yompC gene which resulted from the integration of suicide vector at the yompC locus. In intact cells of Y. enterocolitica, loss of the YompC protein reduced the outer membrane permeability for beta-lactam antibiotics and tetracycline and resulted in a 2-5-fold increase in resistance to these compounds, depending on their chemical properties. Mutation in the ompR regulatory gene resulted in the loss of both YompC and YompF porins, which led to a greater increase of resistance to antibiotics, as compared with the YompC mutant strain. Moreover, the binding assay with HEp-2 cells suggests that YompC may play a role in the adhesion properties of Y. enterocolitica strains.

The level of Yop proteins secreted by Yersinia enterocolitica is changed in maltose mutants.

Enteropathogenic Yersinia enterocolitica strains express a set of plasmid-encoded proteins called Yops, involved in pathogenicity. We studied the influence of the maltose system on the production of Yop proteins and found that the level of Yop proteins of Y. enterocolitica O:9 was reduced in the presence of maltose. Transposon insertion mutants impaired with the maltose transport activity showed a decreased level in the production of Yop proteins. The transcription of the yopH gene for YopH phosphatase in these maltose mutants was unchanged and revealed a maltose mutation impaired in the secretion of Yop proteins instead of their expression.

Physiological properties and production of siderophores detected in Yersinia enterocolitica strain 4-32.

A hydrophilic compound with siderophore activity was isolated from a culture of Yersinia enterocolitica 4-32 grown in an iron-deficient medium. It was found that the siderophore secreted did not belong to the catecholamide and hydroxamate type of siderophores and not yersiniabactin. Supplementation of cultures of Y. enterocolitica 4-32 with sodium chloride (300 mM) resulted in a decrease in the production of siderophores.

Osmoregulation-dependent expression of Yersinia enterocolitica virulence factors.

The effects of hyperosmotic stress on expression of plasmid coded Yop and Yad A proteins--virulence factors of Y.enterocolitica serotype 0:9 were characterized. When cells were shifted to high osmolarity and cultured at 37 degrees C in medium without Ca2+ the production of Yops was inhibited. In contrast, the amount of Yad A protein was unaffected. Addition of glycine betaine to this culture alleviated the effect of high osmolarity. It was also found that hyperosmotic stress causes increased negative supercoiling of DNA in Y. enterocolitica 0:9. Changes in DNA supercoiling coincided with expression of Yop proteins. These results suggest that in high osmolarity the expression of yop genes may be regulated by DNA supercoiling.

Production of Escherichia coli LamB protein in Yersinia enterocolitica.

Plasmid pBCP 68 carrying the lamB gene of Escherichia coli was introduced and expressed in Yersinia enterocolitica cells. The presence of LamB protein in the outer membrane of the wild-type strain of Y. enterocolitica coincided with the loss of the OmpC and OmpF porins. Western blot analysis showed that LamB in Y. enterocolitica cells co-migrated with authentic monomeric LamB, indicating that its signal peptide was recognized and cleaved by Y. enterocolitica and properly integrated into the outer membrane. The expression of LamB made Y. enterocolitica sensitive to phage lambda.

The pho regulon-dependent Ugp uptake system for glycerol-3-phosphate in Escherichia coli is trans inhibited by Pi.

sn-Glycerol-3-phosphate (G3P) or glyceryl phosphoryl phosphodiesters, the substrates of the phoB-dependent Ugp transport system, when transported exclusively through this system, can serve as a sole source of phosphate but not as a sole source of carbon (H. Schweizer, M. Argast, and W. Boos, J. Bacteriol. 150:1154-1163, 1982). In order to explain this phenomenon, we tested two possibilities: repression of the pho regulon by Ugp-mediated transport and feedback inhibition by internal G3P or its degradation product Pi. Using an ugp-lacZ fusion, we found that the expression of ugp does not decline upon exposure to G3P, in contrast to the repressing effect of transport of Pi via the Pst system. This indicated that the Ugp system becomes inhibited after the uptake and metabolism of G3P. Using 32P-labeled G3P, we observed that little Pi is released by cells taking up G3P via the Ugp system but large amounts of Pi are released when the cells are taking up G3P via the GlpT system. Using a glpD mutant that could not oxidize G3P but which could still phosphorylate exogenous glycerol to G3P after GlpF-mediated transport of glycerol, we could not find trans inhibition of Ugp-mediated uptake of exogenous 14C-G3P. However, when allowing uptake of Pi via Pst, we observed a time-dependent inhibition of 14C-G3P taken up by the Ugp transport system. Inhibition was half maximal after 2 min and could be elicited by Pi concentrations below 0.5 mM. Cells had to be starved for Pi in order to observe this inhibition. We conclude that the activity of the Ugp transport system is controlled by the level of internal phosphate.

Maltoporins and maltose-binding proteins of Yersinia enterocolitica.

Two components of the Yersinia enterocolitica maltose transport system, maltoporin (OmpM) and an osmotically shockable periplasmic maltose-binding protein (MBP) were identified. The synthesis of OmpM (apparent Mr 43,000) and transport of maltose into cells of Y. enterocolitica were induced by maltose and maltodextrins. A mutant lacking OmpM was drastically impaired in maltose transport, independent of induction by maltose. The MBP of Y. enterocolitica (apparent Mr 40,000) was found in the osmotic shock fluid. Its synthesis was induced by maltose. Moreover, rabbit antibodies raised against the MBP of E. coli cross-reacted with the analogous protein from Y. enterocolitica. The MBP of Y. enterocolitica restored the maltose transport activities in delta malE mutant cells of E. coli.

Outer membrane permeability and porin proteins of Yersinia enterocolitica.

In intact cells of Yersinia enterocolitica, loss of the YompF protein reduced the outer membrane permeability parameters for two beta-lactam compounds, cephaloridine and nitrocefin. Additional loss of the outer membrane YompC protein resulted in further reductions in the permeability parameters. We conclude that the outer membrane proteins YompC and YompF of Y. enterocolitica are porin proteins.

The OmpC protein of Yersinia enterocolitica: purification and properties.

OmpC, one of the major outer membrane proteins of Yersinia enterocolitica, was isolated and purified to homogeneity. When solubilized at room temperature, this protein appeared on SDS polyacrylamide gel electrophoresis as an oligomer. After heating to the temperature of boiling water, the apparent molecular weight of the monomer was 36,000. The incorporation of purified OmpC into black lipid membranes resulted in an increase in membrane conductance demonstrating pore-forming activity. The reconstituted pores exhibited the characteristics of general diffusion pores. They showed cation selectivity and had a single channel conductance of 1.3 nS in 1.0 M KCl. Assuming a constant diameter of the pore, a length of 6 nm (the width of the outer membrane) and the same ion conductivity inside and outside the pore, the diameter of the pore protein was estimated as 1.0 nm. Polyclonal antibodies were raised against the native, pore-forming protein preparation. These antibodies did not recognize the denatured form of the protein, but cross-reacted with native OmpC and OmpF of Escherichia coli. The regulation of OmpC expression in Y. enterocolitica was dependent on the osmolarity of the medium in the same way as in E. coli.

Outer-membrane permeability to beta-lactam antibiotics in Yersinia enterocolitica.

Two outer-membrane (OM) proteins of Yersinia enterocolitica YOMP-C and YOMP-F appear to function as porins. Mutants that were YOMP-C- and YOMP-F- exhibited changes in cephaloridine and [3H]glucose uptake and increased resistance to beta-lactam antibiotics (especially cephalosporins) and tetracycline. Alterations in OM permeability may contribute to antibiotic resistance in Yersinia.

Penetration of nalidixic acid into Escherichia coli K-12 cells.

Transport of nalidixic acid (NAL) into Escherichia coli cells subjected to osmotic shock, permeabilised with toluene or treated with DNP, CCCP or EDTA, was studied. It was found that osmotic shock and protonophores do not inhibit the transport of [3H]NAL, however, the transport of [3H]DAP and [3H]glucose is reduced. EDTA and toluene enhance penetration of [3H]NAL. This effect is, however, abolished in the presence of Mg++ ions. It is suggested that NAL penetrates into the cell by simple or facilitated diffusion and that the outer membrane of E. coli is the penetration barrier for the drug.

Penetration of nalidixic acid in the cell cycle of Escherichia coli K-12.

Diffusion of nalidixic acid (NAL), DNA synthesis and filamentation of Escherichia coli K-12 cells induced by the drug were investigated in the division cycle. It was found that in nonsynchronous culture and within the range of sublethal NAL concentrations the length of the filaments depends on the dose or the time of action of the drug. In synchronous culture differences are observed in the values of measured parametres between samples from the successive culture phases. Maximum [3H] NAL penetration and DNA synthesis inhibition caused by the drug were noted in the period proceeding directly division. The extent of NAL penetration into the cell in the sample is correlated with the change in length of the NAL-induced filamentous cells.

Mutation affecting resistance of Escherichia coli K12 to nalidixic acid.

A new mutation, nalD, determining resistance of Escherichia coli to nalidixic acid (NAL) is reported. The nalD mutant described is resistant to NAL at 37 degrees C but sensitive at 30 degrees C. It is defective in penetration of NAL and glycerol through the outer membrane at 37 degrees C. The nalD mutation is located half-way between 89 and 89.5 min on the E. coli genetic map.

Influence of penicillin and nalidixic acid on growth and cell division of Escherichia coli K-12.

Ultrastructure of E. coli K-12 cells and the synthesis of DNA in bacteria treated with low concentration of nalidixic acid and penicillin was investigated. In E. coli both drugs caused inhibition of cell division in period D of the life cycle although nalidixic acid inhibits division at an earlier stage of septum formation. The ability of cells to form filaments in the presence of nalidixic acid depends on their age, i.e. time at which cells are taken from synchronous culture.

Effect of various sources of organic carbon and high nitrite and nitrate concentrations on the selection of denitrifying bacteria. I. Stationary cultures.

The effect of methanol, ethanol, acetic acid and glucose together with NaNO2 or KNO3 (1,000 mg N/l) on the intensity of denitrification and selection of denitrifying bacteria from the bottom sludge of nitrogenous wastewater reservoir was examined. Denitrification was found to be the most efficient in medium with ethanol or acetic acid. The presence of glucose facilitated the selection of Alcaligenes faecalis whereas the other carbon sources enabled the selection of bacteria of the genus Pseudomonas: methanol -- P. fluorescens, ethanol -- P. mendocina. In medium with acetic acid species selection depends on the form of nitrogen: NaNO2 -- P. fluorescens, KNO3 -- P. aeruginosa.