Bacterial succession and degradative changes by biofilm on plastic medium for wastewater treatment.

Biofilms contain a diverse range of microorganisms and their varying extracellular polysaccharides. The present study has revealed biofilm succession associated with degradative effects on plastic (polypropylene) and contaminants in sludge. The wet weight of biofilm significantly (p < 0.05) increased; from 0.23 ± 0.01 to 0.44 ± 0.01 g. Similarly, the dry weight of the biofilm increased from 0.02 to 0.05 g. Significant reduction in pathogens (E. coli and feacal coliforms) by MPN technique (>80%) and in chemical parameters (decrease in COD, BOD5 of 73.32 and 69.94%) representing diminution of organic pollutants. Energy dispersive X-ray spectroscopy (EDS) of plastic revealed carbon and oxygen contents, further surface analysis of plastic by scanning electron microscopy (SEM) revealed emergence of profound bacterial growth on the surface. Fourier transform infrared (FTIR) spectroscopy conforms its biotransformation under aerobic conditions after 8 weeks. New peaks developed at the region 1050 and 969 cm(-1) indicating CO and CC bond formation. Thus plastic with 6 weeks old aerobic biofilm (free of pathogens, max. weight, and OD, efficient COD & BOD removal ability) is suggested to be maintained in fixed biofilm reactors for wastewater treatment.

Effect of nutrient limitation on biofilm formation and phosphatase activity of a Citrobacter sp.

A phosphatase-overproducing Citrobacter sp. (NCIMB 40259) was grown in an air-lift reactor in steady-state continuous culture under limitation of carbon, phosphorus or nitrogen. Substantial biofilm formation, and the highest phosphatase activity, were observed under lactose limitation. However, the total amount of biofilm wet biomass and the phosphatase specific activity were reduced in phosphorus- or nitrogen-limited cultures or when glucose was substituted for lactose as the limiting carbon source. Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) showed differences in cell and biofilm morphology in relation to medium composition. Electron microscopy suggested that the differences in biofilm formation may relate to differential expression of fimbriae on the cell surface.

Enzymically mediated bioprecipitation of uranium by a Citrobacter sp. : a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation.

A Citrobacter sp. accumulated uranyl ion (UO2(2+)) via precipitation with phosphate ligand liberated by phosphatase activity. The onset and rate of uranyl phosphate deposition were promoted by NH4(+), forming NH(4)UO(2)PO(4), which has a lower solubility product than NaUO(2)PO(4). This acceleration decoupled the rate-limiting chemical crystallization process from the biochemical phosphate ligand generation. This provided a novel approach to monitor the cell-surface-associated changes using atomic-force microscopy in conjunction with transmission electron microscopy and electron-probe X-ray microanalysis, to visualize deposition of uranyl phosphate at the cell surface. Analysis of extracted surface materials by (31)P NMR spectroscopy showed phosphorus resonances at chemical shifts of 0.3 and 2.0 p.p.m., consistent with monophosphate groups of the lipid A backbone of the lipopolysaccharide (LPS). Addition of fUO2(2+) to the extract gave a yellow precipitate which contained uranyl phosphate, while addition of Cd(2+) gave a chemical shift of both resonances to a single new resonance at 3 p.p.m. Acid-phosphatase-mediated crystal growth exocellularly was suggested by the presence of acid phosphatase, localized by immunogold labelling, on the outer membrane and on material exuded from the cells. Metal deposition is proposed to occur via an initial nucleation with phosphate groups localized within the LPS, shown by other workers to be produced exocellularly in association with phosphatase. The crystals are further consolidated with additional, enzymically generated phosphate in close juxtaposition, giving high loads of LPS-bound uranyl phosphate without loss of activity and distinguishing this from simple biosorption, or periplasmic or cellular metal accumulation mechanisms. Accumulation of 'tethered' metal phosphate within the LPS is suggested to prevent fouling of the cell surface by the accumulated precipitate and localization of phosphatase exocellularly is consistent with its possible functions in homeostatis and metal resistance.

Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme.

A heavy-metal-accumulating Citrobacter sp. has been used for the treatment of metal-laden industrial wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate. A phosphatase-efficient mutant accumulated little UO(2)2+, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate). The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions. Enzyme was also associated with the outer membrane and found extracellularly. Accumulated uranyl phosphate was visible as cell-surface- and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO2PO4.4H2O. Nucleation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation. Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of 'NMR-silent' 112Cd2+. Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate. It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.

Interaction of Citrobacter diversus strains with HEp-2 epithelial and human umbilical vein endothelial cells.

More than 75% of neonates with Citrobacter diversus meningitis develop brain abscesses. Interaction of C. diversus strains with HEp-2 and human umbilical vein endothelial cells (HUVEC) was studied to examine mechanisms related to brain abscess formation. Two of 9 strains invaded HEp-2 cells and 0 of 6 invaded HUVEC better than the others. C. diversus survived at least 20 h within HEp-2 cells (in decreasing numbers). Adhesion to HEp-2 cells was increased in 3 of 4 strains expressing type 1 fimbriae, but this did not correlate with increased invasion. Inhibition of RNA or protein synthesis blocked invasion but not adhesion. Thus, invasion requires ongoing protein synthesis, and adhesion to and invasion of HEp-2 cells by type-1-fimbriated strains are independent steps. Invasion was inhibited by cytochalasin D. A 32-kDa protein found in cerebrospinal fluid isolates of C. diversus was not related to invasion of either cell line. Ability to invade HEp-2 cells was not increased among strains isolated from central nervous system sources.

Uranium bioaccumulation by a Citrobacter sp. as a result of enzymically mediated growth of polycrystalline HUO2PO4.

A Citrobacter sp. accumulates heavy deposits of metal phosphate, derived from an enzymically liberated phosphate ligand. The cells are not subject to saturation constraints and can accumulate several times their own weight of precipitated metal. This high capacity is attributable to biomineralization; uranyl phosphate accumulates as polycrystalline HUO2PO4 at the cell surface. The precipitated metal is indistinguishable from crystalline HUO2PO4.4H2O grown by chemical methods.

Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene.

The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.

Exclusive localization of colicin A in cell cytoplasm of producing bacteria.

The production of colicin A in Citrobacter freundii and in Escherichia coli was studied. After induction with low concentrations of mitomycin C, these organisms differed with regards to cell growth, cell viability, and kinetics of colicin A biosynthesis. Despite these differences, immunoferritin labelling on ultra-thin sections of induced frozen cells demonstrated that colicin A was located exclusively within the cell cytoplasm in both types of bacteria. By using protein markers, it was shown that at no time after induction was colicin A accumulated in the periplasmic space or in inner or outer membranes. These results were confirmed by a biochemical approach. For at least 3 h after induction, colicin A remained associated with producing cells and no colicin A activity was found in the periplasmic space. These results are discussed with reference to the synthesis and export of other bacteriocins.

Effect of glucose on the biochemical properties of the bacterial cytoplasmic membrane.

The cytoplasmic membrane isolated from cells of Citrobacter freundii growing in a cultivation medium with glucose was found to exhibit a decreased ability to oxidize formate and succinate and a decreased activity of formate, succinate and lactate dehydrogenase as compared with the cytoplasmic membrane of cells growing on galactose. The activation energy for dehydrogenation and oxidation of succinate simultaneously increases. The quantitative content of cytochromes and quinones in both types of membranes does not differ considerably. The two types of membranes also do not mutually differ in the qualitative and quantitative representation of fatty acids in lipids and in the sensitivity to the effect of low concentrations of detergents. The mass ratio of proteins and lipids is lower in the membranes of cells grown on glucose.

[Changes in the surface structures of bacteria as a cause of weak immunogenicity of vaccines from several strains of Citrobacter]. / Izmenenie poverkhnostnykh struktur bakterii kak prichina slaboi immunogennosti vaktsin iz nekotorykh shtammov Citrobacter

The authors present the results of studying the weakly immunogenic Citrobacter strains; some O-antigens of Citrobacter (11, 14, 19, 21a, 21b, 26) caused in immunization of rabbits a weak immune response (antibody titre--1:400-1:800). An electron microscopic study of the vaccines (obtained from these strains) and their fragments showed that a partial desquamation and denaturing of the antigenic material occurred in the process of preparation of heated vaccines. Formalin-treated vaccine subjected to preliminary deflaggelation for 2 min at 3000 cps was used for obtaining the O-sera against the mentioned antigens.