The amino acid valine is secreted in continuous-flow bacterial biofilms

Abstract

Biofilms are structured communities characterized by distinctive gene expression patterns and profound physiological changes compared to those of planktonic cultures. Here, we show that many gram-negative bacterial biofilms secrete high levels of a small-molecular-weight compound, which inhibits the growth of only Escherichia coli K-12 and a rare few other natural isolates. We demonstrate both genetically and biochemically that this molecule is the amino acid valine, and we provide evidence that valine production within biofilms results from metabolic changes occurring within high-density biofilm communities when carbon sources are not limiting. This finding identifies a natural environment in which bacteria can encounter high amounts of valine, and we propose that in-biofilm valine secretion may be the long-sought reason for widespread but unexplained valine resistance found in most enterobacteria. Our results experimentally validate the postulated production of metabolites that is characteristic of the conditions associated with some biofilm environments. The identification of such molecules may lead to new approaches for biofilm monitoring and control.

FIG. 1.

Bacteriotoxic effect of the CFT073 in-biofilm supernatant. (A) Growth inhibition activity of CFT073, CFT073ΔkpsD, and CFT073ΔmchB biofilm supernatants on an overlayer of E. coli K-12 cells. (B) Growth curves of MG1655 in the presence of 20% CFT073, CFT073ΔkpsD, and CFT073ΔmchB planktonic supernatant or in-biofilm supernatant. Cultures were performed in M63B1glu. Experiments were performed in duplicate. Error bars represent standard deviations of the means.

FIG. 2.

A high level of valine is present in biofilm supernatant. (A) Inhibitory activity of the CFT073 in-biofilm supernatant (Biofilm-S) on overlays of MG1655, MG1655ΔlivH, and MG1655ilvG⁺ cells. (B) Inhibitory activity of CFT073 in-biofilm supernatant supplemented at a 1:1 ratio with LB medium, nonessential amino acids (NEs; which includes l-alanine; see Materials and Methods), essential amino acids (Es), or the amino acid l-proline, l-valine, l-leucine, or l-isoleucine (all amino acids at a concentration of 50 μg ml⁻¹ each) on an overlayer of MG1655 cells. LB and Es solutions contained l-isoleucine. Ø means that the CFT073 in-biofilm supernatant was not supplemented with exogenous amino acids. (C) The amino acid composition of CFT073 supernatants purified from planktonic culture (Planktonic-S) and biofilm (Biofilm-S). Experiments were performed in triplicate. Error bars represent standard deviations of the means.

FIG. 3.

Isoleucine and valine biosynthesis pathways. (A) ilvBN, ilvGM and ilvIH code for the heterodimeric acetohydroxy acid synthase isozymes AHAS I, AHAS II, and AHAS III, respectively. AHAS I and AHAS III are inhibited in the presence of high amounts of valine, whereas isozyme AHAS II is valine insensitive. Regulations by valine and isoleucine are shown by dashed lines terminated by an arrow for activation or by a vertical line for inhibition. (B) In E. coli K-12, AHAS II is inactivated by a frameshift mutation in ilvG. ilvG sequences of E. coli K-12 and MG1655ilvG⁺ are aligned. The numbers above indicate base numbers from the ATG translation start according to the ilvG⁺ sequence.

FIG. 4.

Effect of the stringent response on valine-related inhibition. Valine-related inhibition on an MG1655 overlayer of (A) the in-biofilm (Biofilm-S) and planktonic (Planktonic-S) supernatants of the CFT073ΔrelA and CFT073ΔrelA ΔspoT mutants and (B) the supernatants of CFT073 cells overproducing ppGpp (pRelA*) and nonoverproducing cells, purified from biofilm (Biofilm-S) or planktonic culture (Planktonic-S).

FIG. 5.

Phenotypic analysis of the planktonic valine-secreting mutants. Valine-related inhibition by supernatants purified from planktonic cultures of CFT073, CFT073ΔsspA, and CFT073ΔtufA (A) and CFT073ΔmchB, CFT073ΔsspA ΔmchB, and CFT073ΔtufA ΔmchB (B) on an overlayer of MG1655 cells. (C) Effect of l-isoleucine addition to the CFT073ΔsspA and the CFT073ΔtufA planktonic supernatant activity on an overlayer of MG1655 cells. (D) Inhibitory activity of the CFT073ΔsspA and the CFT073ΔtufA planktonic supernatants on an overlayer of MG1655ilvG⁺. (E) Analysis of the amino acid composition of the CFT073ΔsspA and the CFT073ΔtufA planktonic supernatants. Experiments were performed in triplicate. Error bars represent standard deviations of the means. (F) Complementation of the CFT073ΔsspA and CFT073ΔtufA mutants with pSspA, pTufA, and the empty plasmid pZE12 as the control.

FIG. 6.

Inactivation of ilvG confers an advantageous phenotype on E. coli K-12. (A) Growth curves of MG1655 and MG1655ilvG⁺ in LB medium at 37°C. (B) Competition experiments: MG1655λattgfp-Km (MG1655) and MG1655λattgfp-amp (MG1655ilvG⁺) cells grown overnight and diluted to an OD of 1 with fresh LB medium were mixed at a 1:1 ratio and grown in LB at 37°C. The population composition was estimated by serial dilutions and plating in order to estimate the CFU of the two strains in the mixed culture. Results are shown as the percentages of colonies of MG1655 and MG1655ilvG⁺ in the mixed culture at 24 h after mixing. Experiments were performed in triplicate. Error bars represent standard deviations of the means.

FIG. 7.

Frequency of MG1655 valine-resistant derivative mutants. MG1655F′ valine-resistant derivatives were isolated from mixed cultures of MG1655F′ and the valine-producing strain CFT073ΔmchBΔkpsD (MGF′/CFT), MG1655F′ and the non-valine-producing strain KS272 (MGF′/KS) or MG1655F′ alone (MGF′/Ø) grown in biofilm or in planktonic culture. Results are expressed as the frequency of valine-resistant mutants of MG1655 isolated after 24 h of growth of the mixed culture. Experiments were performed in triplicate. Error bars represent standard deviations of the means.