Droplet enrichment factors of pigmented and nonpigmented Serratia marcescens: possible selective function for prodigiosin.

Drops produced by bursting bubbles provide a mechanism for the water-to-air transfer and concentration of matter. Bacteria can adsorb to air bubbles rising through bacterial suspensions and enrich the drops formed by the bubbles upon breaking, creating atmospheric biosols which function in dispersal. This bacterial enrichment can be quantified as an enrichment factor (EF), calculated as the ratio of the concentration of bacteria in the drop to that of the bulk bacterial suspension. Bubbles were produced in suspensions of pigmented (prodigiosin-producing) and nonpigmented cultures of Serratia marcescens. EFs for pigmented cultures were greater than EFs for nonpigmented cells. Pigmented cells appeared hydrophobic based on their partitioning in two-phase systems of polyethylene glycol 6000 and dextran T500. The surface hydrophobicity of pigmented cells may result from the hydrophobic nature of prodigiosin and could account for the greater ability of these bacteria to adsorb to air bubbles and enrich airborne droplets. Enhancement of the aerosolization of S. marcescens may be a selective function of the bacterial secondary metabolite prodigiosin.     

Isolation of pigmented and nonpigmented mutants of Serratia marcescens with reduced cell surface hydrophobicity

Enrichment for nonhydrophobic mutants of Serratia marcescens yielded two types: (i) a nonpigmented mutant which exhibited partial hydrophobic characteristics compared with the wild type, as determined by adherence to hexadecane and polystyrene; and (ii) a pigmented, nonhydrophobic mutant whose colonies were translucent with respect to those of the wild type. The data suggest that the pronounced cell surface hydrophobicity of the wild type is mediated by a combination of several surface factors.     

Clinical relevance and virulence factors of pigmented Serratia marcescens

Pigmented Serratia marcescens isolated in a Brazilian hospital were studied with respect to frequency of isolation, serotyping, antibiotic resistance and virulence factors. The serotype most frequent was O6:K14 (53%) and all isolates were resistant to ampicillin, cephalothin and tetracycline. The majority of the isolates (92%) were resistant to the action of human serum and all produced cytotoxins on Vero, CHO, HEp-2 and HeLa cells. These isolates were virulent for mice (LD(50)=10(7) bacteria ml(-1)) and showed virulence factors, but were isolated with low frequency (3. 4%) and caused infection in only 31% of cases. Analysis of serotyping, phage typing and chromosomal DNA revealed at least 13 unrelated strains among pigmented S. marcescens. In conclusion, this work describes a low frequency of isolation of pigmented S. marcescens from clinical specimens, indicating that non-pigmented strains are clinically more significant.     

Scanning electron microscopy of Serratia marcescens producing prodigiosin

Production of high concentrations of prodigiosin by growing cells of Serratia marcescens was accompanied by the formation of extracellular protrusions as was revealed by scanning electron microscopy. Prodigiosin extracted from the bacterium was compared with the extracellular material. Bacteria which did not produce prodigiosin showed no extracellular protrusions.     

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.     

High production of prodigiosin by Serratia marcescens grown on ethanol

Serratia marcescens S389, isolated as an ethanol-utilizing bacterium, produced prodigiosin at up to 3 mg ml^–1 when grown on ethanol and with the omission of inorganic phosphate and NaCl from the medium. This yield was some 200-fold greater than that previously reported.     

Cyclic-AMP inhibition of fimbriae and prodigiosin production by Serratia marcescens is strain-dependent

The cyclic-nucleotide 3′,5′-cyclic AMP (cAMP) is an ancient and widespread regulatory molecule. Previous studies have shown that fimbria production and secondary metabolite production are inhibited by cAMP in the prokaryote Serratia marcescens. This study used genetic manipulations to test the strain specificity of cAMP–cyclic-AMP receptor protein regulation of fimbria production and of the red pigment, prodigiosin. A surprising amount of variation was observed, as multicopy expression of the cAMP-phosphodiesterase gene, cpdS, conferred either an increase or decrease in fimbriae-dependent yeast agglutination and prodigiosin production depending upon the strain background. Mutation of crp, the gene coding for the cAMP-receptor protein, similarly conferred strain-dependent phenotypes. This study shows that three distinct biological properties, modulated by a conserved genetic regulatory molecule, can vary significantly among strains. Such variation can complicate the functional analysis of bacterial phenotypic properties which are dependent upon global genetic regulators such as cAMP.     

Inverse relationship between the flagella formation and prodigiosin synthesis in Serratia marcescens

Treatment by polymyxin B sulfate and ethylenediaminetetraacetate separated a 40 kilodalton (kDa) protein from the nonpigmented Serratia marcescens and even from the nonpigmented bacteria of the pigmented strains, whereas the same treatment separated the 100 kDa protein associated with the pigment formation from the pigmented bacteria. Lysozyme treatment separated the 100 kDa and/or 40 kDa proteins correlated with the pigmented level. The 40 kDa protein was not an outer membrane protein but a flagellin. These results suggest that the flagella formation was inversely related with the pigment formation.     

Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis

Prodigiosin, the bright red pigment produced by many strains of Serratia marcescens, is synthesized by a bifurcated pathway that terminates in the enzymatic condensation of the two final products, a monopyrrole and a bipyrrole . Sau3A fragments of S. marcescens ( Nima ) DNA were introduced into a strain of Escherichia coli K-12 by use of the cosmid vector pHC79 , and transformed clones were selected based on resistance to ampicillin. Among 879 transformants screened, 2 could be induced to synthesize prodigiosin when supplied with either one or both terminal products of the bifurcated pathway. Data are presented to support the idea that production of prodigiosin is not usually mediated by a plasmid.     

The effect of pH on prodigiosin production by non-proliferating cells of Serratia marcescens

The synthesis of prodigiosin by non-proliferating cells of Serratia marcescens was examined at various pH values between 5.5 and 9.5. During incubation in unbuffered medium, pH changed and prodigiosin production was similar regardless of the initial pH. Variations in pigment production were noted when buffers were employed in cultures of non-proliferating cells. The optimum pH for prodigiosin production was 8.0–8.5. Proline oxidase was also measured. The results suggest that the effect of pH may be related to the amount of proline which can be incorporated into prodigiosin.     

Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.     

Adsorptive recovery and purification of prodigiosin from methanol/water solutions of Serratia marcescens fermentation broth

The use of macroporous polymeric adsorption resins for the recovery and purification of prodigiosin from fermentation broth of Serratia marcescens SMDR was systematically studied, in which the broth was pretreated by coagulation with alum and the resulting precipitate was leached by methanol/water solution. Of the seven resins tested, Diaion HP-20 resin was selected considering the adsorption and desorption abilities for prodigiosin at the same time. The optimal compositions of liquid phases for adsorption and desorption were also examined. Batch experiments were performed at 15 ～ 35°C by varying initial prodigiosin concentration in solution (0.05 ～ 0.5 mmol/L), in which molar fraction of each component in the solution was kept constant. The Freundlich and Langmuir equations were used to describe adsorption isotherms and the related thermodynamic functions were also determined. Fixed-bed experiments were finally conducted to obtain the breakthrough characteristics for the adsorption and desorption of prodigiosin. Under optimized conditions, a purification factor of prodigiosin of 11.4 could be obtained from the pretreated broth after one adsorption-desorption run in fixed bed. The present results had demonstrated the promising potential of HP-20 resins for the recovery and purification of prodigiosin from methanol/water solution of Serratia marcescens fermentation broth.