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The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l(-1) h(-1) are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology.

Bacterial pathogenesis requires the precise spatial and temporal control of gene expression, the dynamics of which are controlled by regulatory networks. A network encoded within Salmonella Pathogenicity Island 1 controls the expression of a type III protein secretion system involved in the invasion of host cells. The dynamics of this network are measured in single cells using promoter-green fluorescent protein (gfp) reporters and flow cytometry. During induction, there is a temporal order of gene expression, with transcriptional inputs turning on first, followed by structural and effector genes. The promoters show varying stochastic properties, where graded inputs are converted into all-or-none and hybrid responses. The relaxation dynamics are measured by shifting cells from inducing to noninducing conditions and by measuring fluorescence decay. The gfp expressed from promoters controlling the transcriptional inputs (hilC and hilD) and structural genes (prgH) decay exponentially, with a characteristic time of 50-55 min. In contrast, the gfp expressed from a promoter controlling the expression of effectors (sicA) persists for 110+/-9 min. This promoter is controlled by a genetic circuit, formed by a transcription factor (InvF), a chaperone (SicA), and a secreted protein (SipC), that regulates effector expression in response to the secretion capacity of the cell. A mathematical model of this circuit demonstrates that the delay is due to a split positive feedback loop. This model is tested in a DeltasicA knockout strain, where sicA is complemented with and without the feedback loop. The delay is eliminated when the feedback loop is deleted. Furthermore, a robustness analysis of the model predicts that the delay time can be tuned by changing the affinity of SicA:InvF multimers for an operator in the sicA promoter. This prediction is used to construct a targeted library, which contains mutants with both longer and shorter delays. This combination of theory and experiments provides a platform for predicting how genetic perturbations lead to changes in the global dynamics of a regulatory network.

Organizing heterologous biosyntheses inside bacterial cells can alleviate common problems owing to toxicity, poor kinetic performance, and cofactor imbalances. A subcellular organelle known as a bacterial microcompartment, such as the 1,2-propanediol utilization microcompartment of Salmonella, is a promising chassis for this strategy. Here we demonstrate de novo design of the N-terminal signal sequences used to direct cargo to these microcompartment organelles. We expand the native repertoire of signal sequences using rational and library-based approaches and show that a canonical leucine-zipper motif can function as a signal sequence for microcompartment localization. Our strategy can be applied to generate new signal sequences localizing arbitrary cargo proteins to the 1,2-propanediol utilization microcompartments. [ABSTRACT FROM AUTHOR]

The article discusses the bacterial compartments and its participation on a host-associated lifestyle. Topics discussed include the use of the bacterial compartments by the bacteria for metabolism segregation and the role of gut microbiota on the prevention of host colonization. Also emphasized is the characterization of nutritional immunity for the metal micronutrient transition.

• Pdu MCPs are promising scaffolds for the encapsulation of non-native pathways. • Multiple non-native cargo proteins can be encoded on the genome and co-encapsulated. • Cargo encapsulation levels vary with different tags and genomic loci. • Cargo encapsulation is independent of expression for genomically expressed proteins. Bacterial microcompartments (MCPs) are protein structures that encapsulate specific metabolic pathways in bacteria. The 1,2-propanediol utilization (Pdu) MCP in Salmonella enterica serovar Typhimurium LT2 encapsulates the pathway for 1,2-propanediol degradation to sequester a toxic intermediate, enable cofactor recycling, and enhance pathway flux. The Pdu MCP has potential as an enclosed scaffold for metabolic engineering applications. To successfully use Pdu MCPs for this purpose, however, methods to enable and control heterologous cargo encapsulation are critical. To this end, we here developed a genomic expression platform for cargo encapsulation in Pdu MCPs. We integrated signal sequence-tagged fluorescent reporters into the pdu operon in place of native Pdu enzymes and evaluated the resulting expression and encapsulation levels. We found that fluorescent reporters were successfully co-encapsulated, with varying relative encapsulation levels achieved when using different integration locus and signal sequence combinations. We also observed that the native Pdu signal sequences mediated different encapsulation efficiencies independent of expression levels. This work establishes the genomic integration platform as a viable method for controlling cargo encapsulation, expanding the toolkit toward engineering the Pdu MCP as a tunable nanobioreactor. [ABSTRACT FROM AUTHOR]

Bacterial microcompartments are naturally occurring subcellular organelles of bacteria and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides and represent an important step toward tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity. [ABSTRACT FROM AUTHOR]