Localized Gene Expression in Pseudomonas aeruginosa Biofilms

ABSTRACT Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-β-d-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 μm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-μm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.

Download Full-text

Heterogeneous rpoS and rhlR mRNA Levels and 16S rRNA/rDNA (rRNA Gene) Ratios within Pseudomonas aeruginosa Biofilms, Sampled by Laser Capture Microdissection

Journal of Bacteriology ◽

10.1128/jb.01598-09 ◽

2010 ◽

Vol 192 (12) ◽

pp. 2991-3000 ◽

Cited By ~ 63

Author(s):

Ailyn C. Pérez-Osorio ◽

Kerry S. Williamson ◽

Michael J. Franklin

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

16S Rrna ◽

Stationary Phase ◽

Laser Capture Microdissection ◽

Rrna Gene ◽

Mrna Levels ◽

Bacterial Gene ◽

Laser Capture

ABSTRACT The local environmental conditions in biofilms are dependent on the impinging aqueous solution, chemical diffusion, and the metabolic activities of cells within the biofilms. Chemical gradients established in biofilms lead to physiological heterogeneities in bacterial gene expression. Previously, we used laser capture microdissection (LCM) and quantitative reverse transcription (RT)-PCR to target defined biofilm subpopulations for gene expression studies. Here, we combined this approach with quantitative PCR of bacterial DNA to normalize the amount of gene expression per cell. By comparing the ratio of 16S rRNA to 16S rDNA (rRNA gene), we demonstrated that cells at the top of thick Pseudomonas aeruginosa biofilms have 16S rRNA/genome ratios similar to those of cells in a transition from the exponential phase to the stationary phase. Cells in the middle and bottom layers of these biofilms have ratios that are not significantly different from those of stationary-phase planktonic cultures. Since much of each biofilm appeared to be in a stationary-phase-like state, we analyzed the local amounts of the stationary-phase sigma factor rpoS gene and the quorum-sensing regulator rhlR gene per cell. Surprisingly, the amount of rpoS mRNA was largest at the top of the biofilms at the air-biofilm interface. Less than one rpoS mRNA transcript per cell was observed in the middle or base of the biofilms. The rhlR mRNA content was also greatest at the top of the biofilms, and there was little detectable rhlR expression at the middle or bottom of the biofilms. While the cell density was slightly greater at the bottom of the biofilms, expression of the quorum-sensing regulator occurred primarily at the top of the biofilms, where the cell metabolic activity was greatest, as indicated by local expression of the housekeeping gene acpP and by expression from a constitutive P trc promoter. The results indicate that in thick P. aeruginosa biofilms, cells in the 30 μm adjacent to the air-biofilm interface actively express genes associated with stationary phase, while cells in the interior portions do not express these genes and therefore are in a late-stationary-phase-like state and may be dormant.

Download Full-text

[4] Applications of gene fusions to green fluorescent protein and flow cytometry to the study of bacterial gene expression in host cells

Methods in Enzymology - Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification ◽

10.1016/s0076-6879(00)26046-1 ◽

2000 ◽

pp. 47-73 ◽

Cited By ~ 16

Author(s):

Raphael H. Valdivia ◽

Lalita Ramakrishnan

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Host Cells ◽

Gene Fusions ◽

Bacterial Gene ◽

Bacterial Gene Expression ◽

Green Fluorescent

Download Full-text

Single-Copy Green Fluorescent Protein Gene Fusions Allow Accurate Measurement of Salmonella Gene Expression In Vitro and during Infection of Mammalian Cells

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7480-7491.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7480-7491 ◽

Cited By ~ 172

Author(s):

Isabelle Hautefort ◽

Maria José Proença ◽

Jay C. D. Hinton

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Single Copy ◽

Green Fluorescent Protein Gene ◽

Gene Fusions ◽

Bacterial Gene ◽

Green Fluorescent

ABSTRACT We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as β-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.

Download Full-text

Karyopherin α2: a control step of glucose-sensitive gene expression in hepatic cells

Biochemical Journal ◽

10.1042/bj3640201 ◽

2002 ◽

Vol 364 (1) ◽

pp. 201-209 ◽

Cited By ~ 29

Author(s):

Ghislaine GUILLEMAIN ◽

Maria J. MUÑOZ-ALONSO ◽

Aurélia CASSANY ◽

Martine LOIZEAU ◽

Anne-Marie FAUSSAT ◽

...

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Pyruvate Kinase ◽

Glucose Transporter ◽

Fluorescent Protein ◽

Mrna Levels ◽

Dependent Manner ◽

Mrna Accumulation ◽

Green Fluorescent

Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin α2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin α2, not α1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin α2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein—karyopherin α2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin α2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin α2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin α2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin α2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

Download Full-text