Artem Khlebnikov

Homogeneous expression of the P BAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter

Genes placed under the control of the arabinose-inducible araBAD promoter (P(BAD)) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P(BAD) was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P(BAD) in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P(BAD). In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P(BAD)-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture

The arabinose-inducible promoter P(BAD) is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, the araE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-beta-D-thiogalactopyranoside-inducible P(tac) and P(taclac) promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independent araE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (P(BAD)) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGH mutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.

Effect of lacY expression on homogeneity of induction from the Ptac and Ptrc promoters by natural and synthetic inducers

The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose‐inducible promoters Ptac and Ptrc by the natural inducer lactose and the synthetic inducer isopropyl‐β‐d‐thiogalactopyranoside (IPTG) was investigated. Lactose requires active transport by LacY, whereas IPTG can freely penetrate the cell wall. In E. coli strains lacking a functional LacY, IPTG is required for induction of Ptac and Ptrc. In E. coli strains carrying a functional LacY, induction of Ptrc and Ptac with intermediate concentrations of lactose gave rise to two subpopulations, one fully induced and one uninduced, whereas a single, fully induced population resulted when high inducer concentrations were used. In contrast, induction with IPTG gave rise to a single population of cells at all inducer concentrations in both lacY and lacY + strains.

Modulation of gene expression from the arabinose-inducible araBAD promoter

The arabinose-inducible PBAD promoter suffers from all-or-none gene expression in which cells harboring the natively controlled arabinose transport gene (araE) are either induced or uninduced, the relative fraction of which is controlled by the concentration of arabinose. The population-averaged variation in expression from PBAD as a function of inducer concentration is proportional to the percentage of cells that are fully induced (vs. uninduced) rather than the level of expression in individual cells. Because of its undesirable effects on the expression of heterologous genes, the all-or-none phenomenon was eliminated in Escherichia coli by expression of araE from arabinose-independent (either the Lactococcus lactis constitutive or IPTG-inducible lac) promoters. In these arabinose-transport engineered cells, variation in PBAD expression with arabinose concentration was a result of variation of the expression level in individual cells with all cells in the population having approximately the same induction level. Journal of Industrial Microbiology & Biotechnology (2002) 29, 34–37 doi:10.1038/sj.jim.7000259

Dual Labeling with Green Fluorescent Proteins for Confocal Microscopy

ABSTRACT We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.

Use of soft X-ray microscopy for analysis of early-stage biofilm formation

Soft X-ray microscopy (SXM) using synchrotron radiation is a newly-available technology for high resolution imaging of hydrated specimens, making it potentially valuable for the study of biofilms. In this research, SXM was used to investigate bacterial spatial distribution and biofilm structure during the early stages of colonization of an exposed surface. Pseudomonas putida DMP-1 formed a closely-packed monolayer on a silicon nitride substratum after 24 hours of growth. The initial development of a multilayer, interlocking structure was observed. The soft X-ray images were taken with the XM-1 transmission X-ray microscope, located at the Center for X-ray Optics, Lawrence Berkeley National Laboratory. The results indicate the potential of SXM to provide new insights for the study of biofilms.

Analysis of biofilm structure and gene expression using fluorescence dual labeling

The use of biofilms for the degradation of recalcitrant environmental contaminants or for the production of secondary metabolites necessitates understanding and controlling gene expression. In this work, dual labeling with green fluorescent protein (GFP) variants was used to investigate inducible gene expression in a biofilm. Colocalization of GFP emissions was used to determine regions of attached cells and to correlate structure and activity within the biofilm. The labeling strategy reported here is unique in that the two GFP signals were distinguished by differential excitation rather than differential emission.

A transient mathematical model for maximum respiration activity and oxygen diffusion coefficient estimation in non-steady-state biofilms

A transient mathematical model was established in order to evaluate oxygen diffusivity in non-steady-state biofilms. A submerged fixed bed biofilm system with efficient medium recirculation was investigated for p-toluenesulphonic acid degradation by Comamonas testosteroni T-2 in a multi-species biofilm. Static mixer elements (Sulzer Chemtech Ltd, Switzerland) were used as a support matrix for biofilm formation. Biofilm respiration was estimated using the dynamic gassing-out oxygen uptake method. Based on the dissolved oxygen concentration profiles, the oxygen diffusion coefficient and the maximum respiration activity were calculated. The values of the dissolved oxygen diffusion coefficient varied with biolfilm development and values reported here (2 x 10 -10 -1.2 x 10 -9 m 2 s -1 ) are in good agreement with literature data. Calculated oxygen consumption rates fit well with values obtained in respirometry tests with washed out biofilms. The knowledge of diffusivity changes in biofilms is particularly important for removal capacity estimation and appropriate reactor design.