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.

Intestinal health requires the coexistence of eukaryotic self with the gut microbiota and dysregulated host-microbial interactions can result in intestinal inflammation. Here, we show that colitis improved in T-bet −/− Rag2 −/− mice that consumed a fermented milk product containing Bifidobacterium animalis subsp. lactis DN-173 010 strain. A decrease in cecal pH and alterations in short chain fatty acid profiles occurred with consumption, and there were concomitant increases in the abundance of select lactate-consuming and butyrate-producing bacteria. These metabolic shifts created a nonpermissive environment for the Enterobacteriaceae recently identified as colitogenic in a T-bet −/− Rag2 −/− ulcerative colitis mouse model. In addition, 16S rRNA-based analysis of the T-bet −/− Rag2 −/− fecal microbiota suggest that the structure of the endogenous gut microbiota played a key role in shaping the host response to the bacterial strains studied herein. We have identified features of the gut microbiota, at the membership and functional level, associated with response to this B. lactis -containing fermented milk product, and therefore this model provides a framework for evaluating and optimizing probiotic-based functional foods.

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-␤-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.In 1957, Novick and Weiner (14) studied expression of the lac operon in the presence of inducer concentrations less than that needed for maximal induction (subsaturating concentrations). This early study demonstrated that a fraction of cells in the population was fully induced while the remainder was uninduced and that the number of fully induced cells varied directly with the concentration of inducer. They referred to this mechanism as "all-or-none" or autocatalytic gene expression (14). Autocatalytic gene expression systems contain the genes encoding the transporter under the control of the transported molecule (the inducer). More recently, autocatalytic behavior was also reported for the ara operon (18).Although the all-or-none phenomenon associated with autocatalytic expression systems was demonstrated more than 40 years ago, many of the expression systems currently available continue to be based on similar frameworks and used without regard for this phenomenon. For systems in which population heterogeneity is not important and high-level gene expression is desired, autocatalytic systems remain an ideal choice; expression can be induced to a maximal level in all cells of the populat...

The role of the Escherichia coli lactose permease (LacY) in the homogeneous induction of the lactose-inducible promoters P(tac) and P(trc) by the natural inducer lactose and the synthetic inducer isopropyl-beta-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 P(tac) and P(trc). In E. coli strains carrying a functional LacY, induction of P(trc) and P(tac) 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.