Phenotypic Heterogeneity Affects Stenotrophomonas maltophilia K279a Colony Morphotypes and β-Lactamase Expression

Abstract

Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas maltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the L1 and L2 β-lactamases in response to β-lactam treatment. Here we report that the patient isolate S. maltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bla L1 and bla L2 were transcriptionally the most strongly upregulated genes. Promoter fusions of bla L1 and bla L2 genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla L2 expressing cells as identified by RNA-seq analysis. Overexpression of comE in S. maltophilia K279a reduced the level of cells that were in a bla L2-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including bla L1, bla L2, and comE.

Keywords: K279a; RNA-seq; Stenotrophomonas maltophilia; antibiotic resistance; colony morphotypes; phenotypic heterogeneity; β-lactamases.

FIGURE 1

Phenotypic heterogeneity of SMK279a cells during growth on solid media. (A) SMK279a forms round, uniform colonies when grown without ampicillin on LB agar plates for 48 h at 37°C. (B) Colony heterogeneity on LB agar supplemented with ampicillin showing small and big colonies after growth for 48 h at 37°C. (C) Small colonies (indicated by red dashed circle) and big colonies cultivated on agar plates containing high concentrations of ampicillin (600 μg/ml). (D) Colonies differ slightly in color when cultivated for 48 h on agar plates containing high concentrations of ampicillin (600 μg/ml).

FIGURE 2

Representative scanning electron micrographs (SEM) of SMK279a cells grown in the presence or absence of ampicillin at 37°C for 48 h on LB agar plates. SEM images of SMK279a cells were recorded as previously published (Krohn-Molt et al., 2013). (A) SEM image of cells obtained from colonies cultured in the absence of ampicillin. (B) SEM image of cells obtained from small colonies cultured in the presence of 100 μg/ml ampicillin. (C) SEM image of cells obtained from big colonies cultured in the presence of 100 μg/ml ampicillin. (D) SEM image of cells obtained from big colonies cultured in the presence of 300 μg/ml ampicillin. Cells from (A–C) predominantly formed long filamentous cells (indicated by letter F) and OMVs (indicated by arrows). In the presence of ampicillin, SMK279a cells became enlarged and formed OMVs with sizes up to 677 nm in diameter.

FIGURE 3

RNA-seq and RT-qPCR data analysis for different SMK279a colony morphotypes. (A) Scatter plot of fold-change in transcription of genes among cells of big (Y-axis) and small (X-axis) colonies relative to untreated samples of uniform colonies grown in the absence of ampicillin on LB agar plates. A twofold change in the transcription of genes with adjusted P-value of ≤0.05 is considered as significantly, differentially regulated. Cells forming big colonies differentially regulated several genes mainly involved in degradation of antibiotics. The numbers in the plot indicate most strongly regulated genes: 1, smlt0018; 2, smlt2667; 3, smlt3772; 4, smlt2668; 5, smlt0019; 6, smlt3721; 7, smlt1760; and 7, smlt0596. (B) Differentially regulated genes in small colonies in comparison to big and uniform colonies. Down-regulation of bla_L1 and bla_L2 affected genes involved in regulation, metabolism, virulence, transport and genetic information processing/translation (for details see Table 6). (C) Transcriptome profiles of bla_L2 and the flanking genes ampR (smlt3723) and ampH (smlt3721) among the colony morphotypes. The bla_L2 gene was 6.9-fold and 7.4-fold down-regulated in cells forming small colonies in comparison to big and uniform colonies, respectively. Transcriptome profile images of the leading strand (indicated in red) and the lagging strand (indicated in blue) were generated with the IGB software (Nicol et al., 2009), merged and rearranged on the leading strand for a simplified visualization. (D) RT-qPCR analysis of genes, which were identified in the RNA-seq data set. Expression profiles of five different genes (bla_L2; ampH; bla_L1; smlt0019; smlt0018) were obtained from three independent experiments and analyzed based on the normalized gene expression [2^-ΔΔC(t) method], using rpoD and 16S rRNA as internal reference genes.

FIGURE 4

Analysis of single cell fluorescence of Pbla_L1 and Pbla_L2 promoter gene fusions. (A) Expression of the bla_L1 promoter fused to rfp in SMK279a. Cells were grown at 30°C for 17 h under aerobic conditions (200 rpm) in LB medium containing 100 μg/ml ampicillin. Thereby, 2.0 ± 0.72% cells were in the bla-ON and 98.0 ± 0.72% cells were in the bla-OFF mode. (B) Expression of the bla_L2 promoter fused to rfp in SMK279a grown under the same conditions as indicated in (A). Here, 4.4 ± 0.69% of cells were in the bla-ON and 95.6 ± 0.69% cells were in the bla-OFF mode. (C) Phenotypic heterogeneity observed in cells (carrying the Pbla_L2::rfp fusion) forming long cell chains. Cells were grown overnight under aerobic conditions (120 rpm) in LB medium supplemented with 100 μg/ml ampicillin. (D) Phenotypic heterogeneity observed in cells carrying the Pbla_L2::yfp (E) and the Pbla_L2::cfp promoter gene fusion. (F) Control cells of SMK279a were grown under the same condition as described in (A) carrying a promoterless rfp reporter fusion (P_less::rfp). Images were recorded with 63x/1.30 Oil M27 and 100x/1.30 Oil M27 lenses using a Zeiss Axio Imager 2 fluorescence microscope (Zeiss, Jena, Germany) and employing appropriate filters.

FIGURE 5

Expression of the comE homolog (smlt1134) under its native promoter. (A) Transcriptome profiles of smlt1134 (black arrow) and flanking genes smlt1133; smlt1135 (gray arrows) in an ‘ON’ state, 32 h (blue) and an ‘OFF’ state, 27 h (red). The comE homolog was found to be 5.4-fold down-regulated in the ‘ON’ state (32 h). Transcriptome profile images of the leading strand and the lagging strand [generated with the IGB software (Nicol et al., 2009)] were merged and rearranged on the leading strand for a simplified visualization. (B) Physical map and orientation of the comE homolog and both putative transporter genes in pBBR1MCS-5. The promoter regions were inserted upstream of Pbla_L2::rfp, resulting in pBBR1MCS-5::Pbla_L2::rfp::smlt1134; and pBBR1MCS-5::Pbla_L2::rfp::smlt2851::smlt2852. The recombinant plasmids were transferred to SMK279a cells and challenged with 100 μg/ml ampicillin. (C) Expression of the comE homolog in SMK279a under its native promoter P_ComE. Cells were cultivated for 7 h in LB medium supplemented with 100 μg/ml ampicillin. Expression of comE clearly affects bla_L2 heterogeneous expression resulting in non-fluorescing cells (bla_L2-OFF mode), here less than 1% of cells were in the bla-ON mode. Right and left panels are a bright-field and a fluorescence microscopic image, respectively. (D) Expression of both putative transporter genes (smlt2851; smlt2852) in SMK279a under their native promoter P_2851/2852. Expression of the transporter genes did not alter phenotypic heterogeneous expression of the bla_L2 gene. Here, 5.5 ± 2.12% of the cells were in the bla-ON and 94.5 ± 2.12% cells were in the bla-OFF mode. Cells were cultured under the same condition as indicated in (C). Right and left panels are a bright-field and a fluorescence microscopic image, respectively. Images (C) and (D) were recorded as described in Figure 4.