The quantity and distribution of biofilm growth of Escherichia coli strain ATCC 9723 depends on the carbon/energy source.

Escherichia coli strain 15 (ATCC 9723) formed robust biofilms of two distinct forms on glass tubes. In rich, low-osmolarity medium, the biofilms were restricted to the air/liquid interface, resulting in rings attached to the glass. As it was not evident that these biofilms extended across the liquid surface, we termed them 'ring' rather than 'pellicle' biofilms. In minimal medium supplemented with a non-fermentable substrate as the carbon/energy source, we observed either robust ring biofilms or little biofilm of any type, depending on the substrate. In contrast, fermentable substrates (sugars and sugar derivatives) supported robust biofilms covering most of the solid/liquid interface, which we termed 'tube-covering biofilms'. Maximal biofilm growth was observed when the sugar was a relatively poor substrate, supporting slow growth and known to cause minimal dephosphorylation of regulatory protein Enzyme IIAGlucose of the phosphotransferase system. Compounds found to be inhibitors of biofilm growth, such as lactate, caused a shift from tube-covering to ring form at low concentration and complete loss of biofilm growth at high when added to minimal medium supplemented with a fermentable substrate. Exogenous cAMP activated biofilm growth under all conditions tested, leading to more intense ring or tube-covering biofilms and/or to a shift from ring to tube-covering form.

2-Deoxy-d-glucose is a potent inhibitor of biofilm growth in Escherichia coli.

Escherichia coli strain 15 (ATCC 9723), which forms robust biofilms, was grown under optimal biofilm conditions in NaCl-free Luria-Bertani broth (LB*) or in LB* supplemented with one of the non-metabolizable analogues 2-deoxy-d-glucose (2DG), methyl α-d-mannopyranoside (αMM), or methyl α-d-glucopyranoside (αMG). Biofilm growth was inhibited by mannose analogue 2DG even at very low concentration in unbuffered medium, and the maximal inhibition was enhanced in the presence of either 100 mM KPO4 or 100 mM MOPS, pH 7.5; in buffered medium, concentrations of 0.02 % (1.2 mM) or more inhibited growth nearly completely. In contrast, mannose analogue αMM, which should not be able to enter the cells but has been reported to inhibit biofilm growth by binding to FimH, did not exhibit strong inhibition even at concentrations up to 1.8 % (108 mM). The glucose analogue αMG inhibited biofilm growth, but much less strongly than did 2DG. None of the analogues inhibited planktonic growth or caused a change in pH of the unbuffered medium. Similar inhibitory effects of the analogues were observed in minimal medium. The effects were not strain-specific, as 2DG and αMG also inhibited the weak biofilm growth of E. coli K12.

Biofilm Growth of Escherichia coli Is Subject to cAMP-Dependent and cAMP-Independent Inhibition.

We established that Escherichia coli strain 15 (ATCC 9723) produces both curli and cellulose, and forms robust biofilms. Since this strain is wild type with respect to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), it is an ideal strain in which to investigate the effects of the PTS on the biofilm growth of E. coli. We began by looking into the effects of PTS and non-PTS sugars on the biofilm growth of this strain. All the sugars tested tended to activate biofilm growth at low concentrations but to inhibit biofilm growth at high concentrations. Acidification of the medium was an inhibitory factor in the absence of buffer, but buffering to prevent a pH drop did not prevent the inhibitory effects of the sugars. The concentration at which inhibition set in varied from sugar to sugar. For most sugars, cyclic (c)AMP counteracted the inhibition at the lowest inhibitory concentrations but became ineffective at higher concentrations. Our results suggest that cAMP-dependent catabolite repression, which is mediated by the PTS in E. coli, plays a role in the regulation of biofilm growth in response to sugars. cAMP-independent processes, possibly including Cra, also appear to be involved, in addition to pH effects.

Replacing the general energy-coupling proteins of the phospho-enol-pyruvate: sugar phosphotransferase system of Salmonella typhimurium with fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars.

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EI(Fructose) and FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria-Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12-14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIA(Glucose), leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.

The phosphoenolpyruvate:sugar phosphotransferase system and biofilms in gram-positive bacteria.

This review will examine the connection between the bacterial phosphoenolpyruvate:sugar phosphotransferase system and biofilms. We will consider both the primary role of the phosphoenolpyruvate:sugar phosphotransferase system in sugar uptake by biofilm cells and its possible role in regulatory processes in cells growing as biofilms, and in establishment and maintenance of these biofilms.