Leandro B. Rodríguez-Aparicio

Effect of physical and chemical conditions on the production of colominic acid by Escherichia coli in a defined medium

SummaryA chemically defined medium capable of supporting the growth of Escherichia coli K-235 and optimal for colominic acid (poly-N-acetylneuraminic acid) production has been developed. When the culture was incubated at 37° C for 72 h in 250-ml shake flasks at 250 rpm (in a Gallenkamp incubator IRN-200; orbital diameter 32 mm), the highest polymer level (about 1,350 μg ml-1) was obtained in a minimal medium in which D-xylose and L-proline were the only carbon and nitrogen sources. The optimal pH range for its production (7.6–8.0) was maintained by buffering the medium with 80 mM KH2PO4/Na2HPO4 and the best aeration ratio was 4:1 (volume flask:volume medium). In these conditions high colominic acid biosynthetic activity was detected in the early logarithmic phase and was maximal in 19-h-old cells (A540 nm=4.0). However, the release of polysialic acid into the broth started when growth stopped, showing kinetics typical of a secondary metabolite. The biosynthesis of this polymer was seen to be strictly temperature regulated.

Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: a natural therapeutic strategy useful in the treatment and prevention of skin infections

In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 μg ml−1. Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50–1,000 μg ml−1), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 μg ml−1). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.

Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N‐acetylneuraminic acid (Neu5Ac) with α(2–8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40°C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP‐Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43°C (at least 25 times lower than those observed when the growth temperature was 37°C). The synthesis of these enzymes increased rapidly when bacteria grown at 43°C were transferred to 37°C and decreased dramatically when cells grown at 37°C were transferred to 43°C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with α(2–8) linkages. When the bacterium was grown at 37°C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 μg/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40° no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of N-acetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 μM.

Regulation of capsular polysialic acid biosynthesis by N-acetyl-d-mannosamine, an intermediate of sialic acid metabolism

N‐Acetyl‐d‐mannosamine (ManNAc) is a specific substrate for the synthesis of N‐acetylneuraminic acid, the essential precursor of bacterial capsular polysialic acid (PA). When Escherichia coli K92 used ManNAc as a carbon source, we observed a dramatic reduction (up to 90%) in in vivo PA production. Experiments in which the carbon source was changed revealed that the maximal inhibitory effect occurred when this sugar was present in the medium before the logarithmic phase of bacterial growth had started. Enzymatic analysis revealed that high concentrations of ManNAc‐6‐phosphate inhibit NeuAc lyase, the enzyme that synthesizes NeuAc for PA biosynthesis in E. coli. These results indicate that ManNAc‐6‐phosphate is able to regulate NeuAc lyase activity and modulate the PA synthesis.

Transport of N-acetyl-D-mannosamine and N-acetyl-D-glucosamine in Escherichia coli K1: effect on capsular polysialic acid production

N‐Acetyl‐D‐mannosamine (ManNAc) and N‐acetyl‐D‐glucosamine (GlcNAc) are the essential precursors of N‐acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate‐dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino‐acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.

N-Acetylneuraminic acid uptake in Pasteurella (Mannheimia) haemolytica A2 occurs by an inducible and specific transport system

The N‐acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37°C in 50 mM Tris–HCl buffer, pH 8.0, containing 50 μg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K m for NeuAc was 0.1 μM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N′‐dicyclohexyl‐carbodiimide (50%), and 2,4‐dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.

Fluorometric determination of phenylacetyl-CoA ligase from Pseudomonas putida: A very sensitive assay for a newly described enzyme

Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and Luengo, J.M. (1990) J. Biol. Chem. 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid. This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid. A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase. The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm). The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.

Regulation of colominic acid biosynthesis by temperature: Role of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K‐235 is strictly regulated by temperature. Evidence for the role of cytidine 5′‐monophospho‐N‐acetylneuraminic acid (CMP‐Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP‐Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP‐Neu5Ac necessary for the biosynthesis of colominic acid.

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.