Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a Psychrotrophic Rhodococcus sp.

The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C₁₀ to C₂₁ alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5°C. Mineralization of hexadecane at 5°C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction–gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25°C.     

Identification of alkane hydroxylase genes in Rhodococcus sp. strain TMP2 that degrades a branched alkane

Rhodococcus sp. TMP2 is an alkane-degrading strain that can grow with a branched alkane as a sole carbon source. TMP2 degrades considerable amounts of pristane at 20 degrees C but not at 30 degrees C. In order to gain insights into microbial alkane degradation, we characterized one of the key enzymes for alkane degradation. TMP2 contains at least five genes for membrane-bound, non-heme iron, alkane hydroxylase, known as AlkB (alkB1-5). Phylogenetical analysis using bacterial alkB genes indicates that TMP2 is a close relative of the alkane-degrading bacteria, such as Rhodococcus erythropolis NRRL B-16531 and Q15. RT-PCR analysis showed that expressions of the genes for AlkB1 and AlkB2 were apparently induced by the addition of pristane at a low temperature. The results suggest that TMP2 recruits certain alkane hydroxylase systems to utilize a branched alkane under low temperature conditions.     

Identification and characterization of novel low-temperature-inducible promoters of Escherichia coli.

Escherichia coli promoters that are more active at low temperature (15 to 20 degrees C) than at 37 degrees C were identified by using the transposon Tn5-lac to generate promoter fusions expressing beta-galactosidase (beta-Gal). Tn5-lac insertions that resulted in low-temperature-regulated beta-Gal expression were isolated by selecting kanamycin-resistant mutants capable of growth on lactose minimal medium at 15 degrees C but which grew poorly at 37 degrees C on this medium. Seven independent mutants were selected for further studies. In one such strain, designated WQ11, a temperature shift from 37 degrees C to either 20 or 15 degrees C resulted in a 15- to 24-fold induction of beta-Gal expression. Extended growth at 20 or 15 degrees C resulted in 36- to 42-fold-higher beta-Gal expression over that of cells grown at 37 degrees C. Treatment of WQ11 with streptomycin, reported to induce a response similar to heat shock, failed to induce beta-Gal expression. In contrast, treatment with either chloramphenicol or tetracycline, which mimics a cold shock response, resulted in a fourfold induction of beta-Gal expression in strain WQ11. Hfr genetic mapping studies complemented by physical mapping indicated that in at least three mutants (WQ3, WQ6, and WQ11), Tn5-lac insertions mapped at unique sites where no known cold shock genes have been reported. The Tn5-lac insertions of these mutants mapped to 81, 12, and 34 min on the E. coli chromosome, respectively. The cold-inducible promoters from two of the mutants (WQ3 and WQ11) were cloned and sequenced, and their temperature regulation was examined. Comparison of the nucleotide sequences of these two promoters with the regulatory elements of other known cold shock genes identified the sequence CCAAT as a putative conserved motif.     

The Marine Isolate <Emphasis Type="Italic">Novosphingobium</Emphasis> sp. PP1Y Shows Specific Adaptation to Use the Aromatic Fraction of Fuels as the Sole Carbon and Energy Source

Novosphingobium sp. PP1Y, isolated from a surface seawater sample collected from a closed bay in the harbour of Pozzuoli (Naples, Italy), uses fuels as its sole carbon and energy source. Like some other Sphingomonads, this strain can grow as either planktonic free cells or sessile-aggregated flocks. In addition, this strain was found to grow as biofilm on several types of solid and liquid hydrophobic surfaces including polystyrene, polypropylene and diesel oil. Strain PP1Y is not able to grow on pure alkanes or alkane mixtures but is able to grow on a surprisingly wide range of aromatic compounds including mono, bi, tri and tetracyclic aromatic hydrocarbons and heterocyclic compounds. During growth on diesel oil, the organic layer is emulsified resulting in the formation of small biofilm-coated drops, whereas during growth on aromatic hydrocarbons dissolved in paraffin the oil layer is emulsified but the drops are coated only if the mixtures contain selected aromatic compounds, like pyrene, propylbenzene, tetrahydronaphthalene and heterocyclic compounds. These peculiar characteristics suggest strain PP1Y has adapted to efficiently grow at the water/fuel interface using the aromatic fraction of fuels as the sole carbon and energy source.     

Initial reactions in anaerobic alkane degradation by a sulfate reducer, strain AK-01

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-(13)C(2)]hexadecane or perdeuterated pentadecane was used as the growth substrate, (13)C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the (13)C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two (13)C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1, 2-(13)C(2)]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.     

An antarctic psychrotrophic bacterium Halomonas sp. ANT-3b, growing on n-hexadecane, produces a new emulsyfying glycolipid

A bacterial strain ANT-3b was isolated at the sea-ice seawater interface from Terra Nova Bay station, Ross Sea, Antarctica. It was isolated on mineral medium supplemented with 2% diesel fuel as a sole carbon and energy source and grown routinely on 2% n-hexadecane. Analysis of 16S rRNA gene sequence indicates that the strain has 99.8% sequence similarity with Halomonas neptunia. The strain ANT-3b was grown in mineral medium supplemented with n-hexadecane between 4 and 20 degrees C, but not at 30 degrees C. The maximum degradation rate of the n-alkane was measured at 15 degrees C, with 5.6+/-1.7 mg O2 microg(-1) protein d(-1). The strain ANT-3b produced emulsifying compounds when grown on n-hexadecane, but not on mineral medium supplemented with D-fructose. A preliminary characterisation of the emulsifier was carried out. The lipid moiety contained a mixture of fatty acids with a following composition in molar ratio: caprylic acid 18.85, myristic acid 1.0, palmitic acid 9.68, palmitoleic acid 5.69 and oleic acid 1.26. The polysaccharide moiety also contained a mixture of sugars with the following molar ratio: mannose 1.71, galactose 1.00 and glucose 2.96. The molecular weight of the glycolipid component determined by gel permeation chromatography was in the 18 kDa range and contained smaller fragments, possibly oligomeric contaminants. Transmission electron microscopy showed contact between the glycolipid secreted by the strain and n-hexadecane broken down to nanodroplets at the water interface, to form a material with mesophase (liquid crystal) organisation.     

The effect of temperature shifts on protein synthesis by the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the temperature-related characteristics of protein synthesis in cells grown at 0 degrees C differed from those of cells grown at 13 degrees C. Cells grown at 0 degrees C and 13 degrees C transported amino acids at the same rates, dependent on the temperature at which rates were measured. The rates of protein synthesis in extracts of cells grown at 0 degrees C and at 13 degrees C differed, as a result of the changes in the properties of the soluble fraction involved in protein synthesis. Concurrently, levels of more than 24 polypeptides in the soluble fraction changed considerably. These results suggest that the difference in temperature dependence of protein synthesis in cells grown at various temperatures may be brought about by specific changes in the levels of a small number of polypeptides (less than 15% of the total number of proteins detected by silver-staining) in response to a change in temperature.     

Identification of alkane monoxygenase genes inAcinetobacter venetianus VE-C3 and analysis of mutants impaired in diesel fuel degradation

Cells ofAcinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process inA. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis ofalkM andrubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk^? mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and energy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genetic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be similar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also.     

Effects of incubation temperature on growth and production of exopolysaccharides by an antarctic sea ice bacterium grown in batch culture

The sea ice microbial community plays a key role in the productivity of the Southern Ocean. Exopolysaccharide (EPS) is a major component of the exopolymer secreted by many marine bacteria to enhance survival and is abundant in sea ice brine channels, but little is known about its function there. This study investigated the effects of temperature on EPS production in batch culture by CAM025, a marine bacterium isolated from sea ice sampled from the Southern Ocean. Previous studies have shown that CAM025 is a member of the genus Pseudoalteromonas and therefore belongs to a group found to be abundant in sea ice by culture-dependent and -independent techniques. Batch cultures were grown at -2 degrees C, 10 degrees C, and 20 degrees C, and cell number, optical density, pH, glucose concentration, and viscosity were monitored. The yield of EPS at -2 degrees C and 10 degrees C was 30 times higher than at 20 degrees C, which is the optimum growth temperature for many psychrotolerant strains. EPS may have a cryoprotective role in brine channels of sea ice, where extremes of high salinity and low temperature impose pressures on microbial growth and survival. The EPS produced at -2 degrees C and 10 degrees C had a higher uronic acid content than that produced at 20 degrees C. The availability of iron as a trace metal is of critical importance in the Southern Ocean, where it is known to limit primary production. EPS from strain CAM025 is polyanionic and may bind dissolved cations such at trace metals, and therefore the presence of bacterial EPS in the Antarctic marine environment may have important ecological implications.     

Stimulation of Lipase Production During Bacterial Growth on Alkanes

Acinetobacter lwoffi strain O₁₆, a facultative psychrophile, can grow on crude oil, hexadecane, octadecane, and most alkanes when tested at 20 but not at 30°C. Growth occurred on a few alkanes at 30°C but after a longer lag than at 20°C. Cells grown on alkanes as sole carbon sources had high levels of cell-bound lipase. In contrast, previous work has shown that those grown on complex medium produced cell-free lipase and those grown on defined medium without alkanes produced little or no lipase. Low concentrations of the detergent Triton X-100 caused the liberation of most of the lipase activity of alkane-grown cells and increased total lipase activity. When ethanol and hexadecane were both present in a mineral medium, diauxic growth occurred; until the ethanol was completely used up, hexadecane was not utilized, and the lipase activity was very low. When growth on hexadecane began, lipase activity increased, reaching a level 50- to 100-fold higher than that of cells growing on ethanol. A similar pattern of lipase formation and hexadecane utilization was observed with Pseudomonas aeruginosa. Whenever A. lwoffi and other bacteria degraded alkanes they exhibited substantial lipase activity. Not all bacteria that produced lipase, however, could attack alkanes. Bacteria that could not produce lipase did not attack alkanes. The results suggest that a correlation may exist between lipase formation and alkane utilization.     

Desulfobacter psychrotolerans sp. nov., a new psychrotolerant sulfate-reducing bacterium and descriptions of its physiological response to temperature changes

A psychrotrolerant acetate-oxidizing sulfate-reducing bacterium (strain akvb(T)) was isolated from sediment from the northern part of The North Sea with annual temperature fluctuations between 8 and 14 degrees C. Of the various substrates tested, strain akvb(T) grew exclusively by the oxidation of acetate coupled to the reduction of sulfate. The cells were motile, thick rods with round ends and grew in dense aggregates. Strain akvb(T) grew at temperatures ranging from -3.6 to 26.3 degrees C. Optimal growth was observed at 20 degrees C. The highest cell specific sulfate reduction rate of 6.2 fmol cell(-1) d(-1) determined by the (35)SO(2-)(40) method was measured at 26 degrees C. The temperature range of short-term sulfate reduction rates exceeded the temperature range of growth by 5 degrees C. The Arrhenius relationship for the temperature dependence of growth and sulfate reduction was linear, with two distinct slopes below the optimum temperatures of both processes. The critical temperature was 6.4 degrees C. The highest growth yield (4.3-4.5 g dry weight mol(-1) acetate) was determined at temperatures between 5 and 15 degrees C. The cellular fatty acid composition was determined with cultures grown at 4 and 20 degrees C, respectively. The relative proportion of cellular unsaturated fatty acids (e.g. 16:1omega7c) was higher in cells grown at 4 degrees C than in cells grown at 20 degrees C. The physiological responses to temperature changes showed that strain akvb(T) was well adapted to the temperature regime of the environment from which it was isolated. Phylogenetic analysis showed that strain akvb(T) is closest related to Desulfobacter hydrogenophilus, with a 16S rRNA gene sequence similarity of 98.6%. DNA-DNA-hybridization showed a similarity of 32% between D. hydrogenophilus and strain akvb(T). Based on phenotypic and DNA-based characteristics we propose that strain akvb(T) is a member of a new species, Desulfobacter psychrotolerans sp. nov.     

Morphology of an Escherichia coli mutant with a temperature-dependent round cell shape.

Mutants of Escherichia coli capable of growing in the presence of 10 microgram of mecillinam per ml were selected after intensive mutagenesis. Of these mutants, 1.4% formed normal, rod-shaped cells at 30 degrees C but grew as spherical cells at 42 degrees C. The phenotype of one of these rod(Ts) mutants was 88% cotransducible with lip (14.3 min), and all lip+ rod(Ts) transductants of a lip recipient had the following characteristics: (i) growth was relatively sensitive to mecillinam at 30 degrees C but relatively resistant to mecillinam at 42 degrees C; (ii) penicillin-binding protein 2 was present in membranes of cells grown at 30 degrees C in reduced amounts and was undetectable in the membranes of cells grown at 42 degrees C. The mecillinam resistance, penicillin-binding protein 2 defect, and rod phenotypes all cotransduced with lip with high frequency. Thus the mutation [rodA(Ts)] is most likely in the gene for penicillin-binding protein 2 and causes the organism to grow as a sphere at 42 degrees C, although it grows with normal rodlike morphology at 30 degrees C. At 42 degrees C, cells of this strain were round with many wrinkles on their surfaces, as revealed by scanning electron microscopy. In these round cells, chromosomes were dispersed or distributed peripherally, in contrast to normal rod-shaped cells which had centrally located, more condensed chromosomes. The round cells divided asymmetrically on solid agar, and it seemed that the plane of each successive division was perpendicular to the preceding one. On temperature shift-down in liquid medium many cells with abnormal morphology appeared before normal rod-shaped cells developed. Few abnormal cells were seen when cells were placed on solid medium during temperature shift-down. These pleiotropic effects are presumably caused by one or more mutations in the rodA gene.     

Initial Reactions in Anaerobic Alkane Degradation by a Sulfate Reducer, Strain AK-01

An alkane-degrading, sulfate-reducing bacterial strain, AK-01, isolated from a petroleum-contaminated sediment was studied to elucidate its mechanism of alkane metabolism. Total cellular fatty acids of AK-01 were predominantly C even when it was grown on C-even alkanes and were predominantly C odd when grown on C-odd alkanes, suggesting that the bacterium anaerobically oxidizes alkanes to fatty acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty acids were specifically found only when AK-01 was grown on alkanes, and their chain lengths always correlated with those of the alkanes. When [1,2-¹³C₂]hexadecane or perdeuterated pentadecane was used as the growth substrate, ¹³C-labeled 2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were alkane derived. Examination of the ¹³C-labeled 2-, 4-, and 6-methylated fatty acids by mass spectrometry showed that each of them contained two ¹³C atoms, located at the methyl group and the adjacent carbon, thus indicating that the methyl group was the original terminal carbon of the [1,2-¹³C₂]hexadecane. For perdeuterated pentadecane, the presence of three deuterium atoms, on the methyl group and its adjacent carbon, in each of the deuterated 2-, 4-, and 6-methylated fatty acids further supported the hypothesis that the methyl group was the terminal carbon of the alkane. Thus, exogenous carbon appears to be initially added to an alkane subterminally at the C-2 position such that the original terminal carbon of the alkane becomes a methyl group on the subsequently formed fatty acid. The carbon addition reaction, however, does not appear to be a direct carboxylation of inorganic bicarbonate. A pathway for anaerobic metabolism of alkanes by strain AK-01 is proposed.     

Effects of the temperature range and the lack of beta-ketoacyl acyl-carrier protein synthase II on fatty acid synthesis in Escherichia coli K12 after shifts in temperature

Escherichia coli K12 cells grown at higher temperatures and then subjected to lower temperatures produce fatty acids with higher unsaturated/saturated ratios than cells completely adapted to the lower temperatures (Okuyama et al. (1982) J. Biol. Chem. 257, 4812-4817). This hyper-response was not an artefact of chloramphenicol treatment and was observed when the shift-down was more than 20 degrees C in the cells grown at either 40 degrees C or 35 degrees C. In contrast, cells grown at either 25 degrees C or 30 degrees C showed no appreciable hyper-response in terms of unsaturated/saturated ratio on temperature shifts to as low as 10 degrees C. By combining shift-down and shift-up experiments, we could show the presence of different types of temperature dependency in the fatty acid-synthesizing systems of cells grown at various temperatures. Contrary to wild-type cells which synthesized mainly cis-vaccenate on down-shift to 10 degrees C, a mutant strain lacking beta-ketoacyl acyl-carrier protein synthase II synthesized more palmitoleate (16:1) and less palmitate at 10 degrees C than at 40 degrees C. The average chain lengths of saturated and unsaturated fatty acids also changed, but differently, between the mutant and wild-type cells on shifts of temperature. Thus, the mutant strain has a temperature-dependent fatty acid-synthesizing system qualitatively different from that seen in a wild-type strain.     

Variation in the structure and bacteriophage-inactivating capacity of Salmonella anatum lipopolysaccharide as a function of growth temperature.

Growth temperature affects both the structure and the phage-inactivating capacity of Salmonella anatum A1 lipopolysaccharide. Whereas S. anatum cells normally synthesize smooth lipopolysaccharide when grown at physiological temperature (37 degrees C), a partial smooth-rough transition occurs when cells are grown at low temperature (20 to 25 degrees C). The synthesis at low growth temperature of lipopolysaccharide molecules lacking O-antigen was detected both by increased sensitivity of cells to the rough-specific bacteriophage Felix O-1 and by fractionation of oligosaccharides derived from lipopolysaccharide by mild acid hydrolysis. Growth temperature-induced changes in the structure of S. anatum A1 lipopolysaccharide also affected its ability to inactivate epsilon15, a bacteriophage that binds initially to the O-antigen portion of the molecule. Purified lipopolysaccharide prepared from cells grown at low growth temperature exhibited a higher in vitro phage-inactivating capacity than did lipopolysaccharide prepared from cells grown at physiological temperature (37 degrees C).     

Structural analysis of mucoidan, an acidic extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides (EPS), which are assumed to play an important role in the hydrocarbon tolerance of R. erythropolis PR4. The strain produced an acidic EPS, mucoidan, together with a fatty acid-containing EPS, PR4 FACEPS. The chemical structure of the mucoidan was determined using (1)H and (13)C NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The mucoidan was shown to consist of a pentasaccharide repeating unit with the following structure: [structure: see text].     

Phase separations in membranes of Anacystis nidulans grown at different temperatures.

Freeze fracture electron microscopy studies were performed on samples of Anacystis nidulans quenched from different temperatures. Membrane lipid phase separations were observed to take place over the ranges 15--30 degrees C, 5--25 degrees C and -5--15 degrees C for cultures grown at 38, 28 and 18 degrees C, respectively. Differential scanning calorimetry heating curves showed endotherms which coincided with these temperature ranges. Variations of phase separation temperatures with growth temperature, and hysteresis effects in the calorimetric measurements, were related to changes in the fatty acid composition of membrane lipids.     

Listeria monocytogenes LO28: surface physicochemical properties and ability to form biofilms at different temperatures and growth phases

The surface physicochemical properties of Listeria monocytogenes LO28 under different conditions (temperature and growth phase) were determined by use of microelectrophoresis and microbial adhesion to solvents. The effect of these parameters on adhesion and biofilm formation by L. monocytogenes LO28 on hydrophilic (stainless steel) and hydrophobic (polytetrafluoroethylene [PTFE]) surfaces was assessed. The bacterial cells were always negatively charged and possessed hydrophilic surface properties, which were negatively correlated with growth temperature. The colonization of the two surfaces, monitored by scanning electron microscopy, epifluorescence microscopy, and cell enumeration, showed that the strain had a great capacity to colonize both surfaces whatever the incubation temperature. However, biofilm formation was faster on the hydrophilic substratum. After 5 days at 37 or 20 degrees C, the biofilm structure was composed of aggregates with a three-dimensional shape, but significant detachment took place on PTFE at 37 degrees C. At 8 degrees C, only a bacterial monolayer was visible on stainless steel, while no growth was observed on PTFE. The growth phase of bacteria used to inoculate surfaces had a significant effect only in some cases during the first steps of biofilm formation. The surface physicochemical properties of the strain are correlated with adhesion and surface colonization.     

Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar Typhimurium DT104

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37 degrees C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37 degrees C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25 degrees C than at 37 degrees C. Since the CP was shown to be produced at both 37 degrees C and 25 degrees C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.