Galactose metabolism plays a crucial role in biofilm formation by Bacillus subtilis

Abstract

Galactose is a common monosaccharide that can be utilized by all living organisms via the activities of three main enzymes that make up the Leloir pathway: GalK, GalT, and GalE. In Bacillus subtilis, the absence of GalE causes sensitivity to exogenous galactose, leading to rapid cell lysis. This effect can be attributed to the accumulation of toxic galactose metabolites, since the galE mutant is blocked in the final step of galactose catabolism. In a screen for suppressor mutants restoring viability to a galE null mutant in the presence of galactose, we identified mutations in sinR, which is the major biofilm repressor gene. These mutations caused an increase in the production of the exopolysaccharide (EPS) component of the biofilm matrix. We propose that UDP-galactose is the toxic galactose metabolite and that it is used in the synthesis of EPS. Thus, EPS production can function as a shunt mechanism for this toxic molecule. Additionally, we demonstrated that galactose metabolism genes play an essential role in B. subtilis biofilm formation and that the expressions of both the gal and eps genes are interrelated. Finally, we propose that B. subtilis and other members of the Bacillus genus may have evolved to utilize naturally occurring polymers of galactose, such as galactan, as carbon sources.

Importance: Bacteria switch from unicellular to multicellular states by producing extracellular matrices that contain exopolysaccharides. In such aggregates, known as biofilms, bacteria are more resistant to antibiotics. This makes biofilms a serious problem in clinical settings. The resilience of biofilms makes them very useful in industrial settings. Thus, understanding the production of biofilm matrices is an important problem in microbiology. In studying the synthesis of the biofilm matrix of Bacillus subtilis, we provide further understanding of a long-standing microbiological observation that certain mutants defective in the utilization of galactose became sensitive to it. In this work, we show that the toxicity observed before was because cells were grown under conditions that were not propitious to produce the exopolysaccharide component of the matrix. When cells are grown under conditions that favor matrix production, the toxicity of galactose is relieved. This allowed us to demonstrate that galactose metabolism is essential for the synthesis of the extracellular matrix.

FIG 1

Galactose toxicity-induced cell lysis in the galE mutant by galactose metabolites. (A) Leloir pathway for galactose metabolism in bacteria. galK encodes a galactose kinase, galT encodes a galactose-1-phosphate uridyltransferase, and galE encodes a UDP-galactose epimerase. Glc, glucose; Gal, galactose. (B) Growth of the wild type (3610) (WT) and the ΔgalE mutant (FC300) in LB (Luria-Bertani) medium in the presence or absence of 0.5% galactose. Arrows indicate the time point at which cell samples were collected for microscopy analyses (results shown in panel C). (C) Live/dead cell staining of the B. subtilis ΔgalE mutant (FC300). Cells were grown in LB medium with or without supplementation of 0.5% galactose and collected 3 h after inoculation (indicated by arrows in panel B). Cells were treated with dyes for live/dead cell staining and examined under fluorescence microscopy. Arrows points to cells showing distinct bulge-like structures. (D) Growth of the wild type (3610) and ΔgalE mutant (FC300) of B. subtilis in LB medium supplemented or not supplemented with 0.5% galactose (Gal) or 0.5% d-oxy-galactose (d-oxy-Gal). (E) Growth of the ΔgalETK (YC563), ΔgalETK amyE::galK (YC810), and ΔgalETK amyE::galTK (YC811) mutants in LB medium supplemented or not supplemented with 0.5% galactose (Gal).

FIG 2

Producing EPS (exopolysaccharide) as a shunt pathway for toxic metabolites from galactose. (A to C) Characterization of galE suppressor mutants resistant to galactose toxicity. Cells were inoculated on LB agar plates supplemented with 0.5% galactose. (D) Overexpression of the epsA-O operon suppressed galactose toxicity in the galE mutant. galE::tet P_hyspank-epsA-O cells (YC776) were grown in MSgg media in the absence (left) or presence (right) of 0.1% galactose and with various amounts of IPTG (see the key). The galE epsA-O double mutant is also included as a control. (E) Sugar composition analysis of EPS from wild-type (3610) or ΔepsA-O mutant (YC771) pellicles. Glc, glucose; GalNAc, N-acetyl-galactose; Gal, galactose.

FIG 3

The gal genes are important for biofilm formation. Top-down view of pellicle formation of the wild type (3610) and the ΔgalE (FC300), ΔgalTK (YC532), and ΔgalE ΔgalTK (YC563) mutants in MSgg medium with or without 0.5% galactose.

FIG 4

Regulation of the gal genes in B. subtilis. (A) β-Galactosidase activities of the wild type (YC777), the sinI mutant (YC778), and the spo0A mutant (YC779), which harbor the P_galE-lacZ reporter at the amyE locus on the chromosome. Cells were grown in MSgg shaking culture, and hour 5 postinoculation was approximately the start of the stationary phase. (B) Genetic organization of the ywcC-gtcA-galK-galT gene cluster in B. subtilis. (C) Results of the reverse transcription-PCR (RT-PCR) using primers that amplify part of the mRNA transcript of the ywcC-gtcA-galK-galT gene cluster. The amplified region (about 3 kb) is indicated by the horizontal double-headed arrow in panel B. PCR amplification without reverse transcription (−RT) was used as a negative control. (D) β-Galactosidase activities of the wild type (YC783) and the ywcC mutant (YC784), which contain the P_ywcC-lacZ reporter at the chromosomal amyE locus. (E) Proposed model for the concerted regulation of the epsA-O operon and the gal genes in B. subtilis. Under normal biofilm-inducing conditions, the master regulator Spo0A activates both sinI, which leads to derepression of the epsA-O operon, and galE, whose protein product generates UDP-Gal as a key nucleotide-sugar substrate for EPS biosynthesis. Under alternative conditions, a TetR-like regulator, YwcC, derepresses both slrA, which also leads to derepression of the epsA-O operon and galK and galT in the same operon, whose protein products convert exogenous galactose to UDP-Gal.

FIG 5

Utilization of galactan by B. subtilis. (A) Genetic organization of the putative gene cluster for galactan utilization in B. subtilis. ganB encodes a putative endo-1,4-beta-galactosidase, ganA encodes a putative β-galactosidase, cycB-ganP-ganQ encodes a sugar transportation system, and lacR encodes a LacI family transcription repressor. (B) Growth of the wild type (3610), ΔcycB-ganPQAB mutant (YC1063), ΔgalE mutant (FC300), and ΔgalKT mutant (YC532) in modified M9 minimal medium with 0.5% galactan as the sole carbon source. As a control, these strains were also grown in M9 minimal medium with 0.5% glucose as the sole carbon source. (C) Strains were grown in LB medium (lower left) or LB medium supplemented with 0.5% galactan (lower right) to test galactan toxicity. Strains are designated as follows: WT is strain 3610, ΔcycB-ganPQAB is YC1063, ΔgalE is FC300, and ΔcycB-ganPQAB ΔgalE is YC1095.

FIG 6

Galactan metabolism in B. subtilis and other bacteria. (A) Proposed pathway in B. subtilis for utilization of naturally occurring galactan. Galactan is first broken down to galactotriose by GanB outside the cell. Galactotriose is then taken up by the permease composed of CycB-GanP-GanQ and further broken down to galactose by GanA. Galactose is eventually converted to glucose by the enzymes in the Leloir pathway encoded by the gal genes or becomes a sugar substrate of EPS during biofilm formation. (B) Genetic arrangements of the putative gene clusters for utilization of galactan in various bacteria. In two plant-associated Bacillus species, B. licheniformis and B. amyloliquefaciens, as well as in a gut-associated bacterium, Lactobacillus acidophilus, the homologous (to cycB-ganPQAB in B. subtilis) genes whose protein products are predicted to function in the breakdown of galactan to galactose are not only clustered but also grouped with the gal genes whose protein products are involved in further metabolism of galactose. Conservation of this genetic arrangement of the above-described genes in various bacteria implies that these bacteria adapted to utilize host-derived sugar polymers. In L. acidophilus, the gene cluster contains three ganA paralogs (light green) and two unknown phage-like genes (gray). In B. amyloliquefaciens, the two permease-like genes (blue) encode a putative PtII sugar uptake system, whereas in L. acidophilus, a single lacS gene (blue) encodes a putative sugar permease.