Multiomics Study of Bacterial Growth Arrest in a Synthetic Biology Application.

We investigated the scalability of a previously developed growth switch based on external control of RNA polymerase expression. Our results indicate that, in liter-scale bioreactors operating in fed-batch mode, growth-arrested Escherichia coli cells are able to convert glucose to glycerol at an increased yield. A multiomics quantification of the physiology of the cells shows that, apart from acetate production, few metabolic side effects occur. However, a number of specific responses to growth slow-down and growth arrest are launched at the transcriptional level. These notably include the downregulation of genes involved in growth-associated processes, such as amino acid and nucleotide metabolism and translation. Interestingly, the transcriptional responses are buffered at the proteome level, probably due to the strong decrease of the total mRNA concentration after the diminution of transcriptional activity and the absence of growth dilution of proteins. Growth arrest thus reduces the opportunities for dynamically adjusting the proteome composition, which poses constraints on the design of biotechnological production processes but may also avoid the initiation of deleterious stress responses.

The C-terminal amphipathic alpha-helix of Pseudomonas aeruginosa PelC outer membrane protein is required for its function.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes numerous infections and can adopt a versatile lifestyle. During chronic infection, P. aeruginosa becomes established as a bacterial community known as a biofilm. Biofilm formation results from the production of a matrix mainly comprised of exopolysaccharides. P. aeruginosa possesses several gene clusters which contribute to the formation of the matrix, including the pel genes. Among the pel genes, pelC encodes an outer membrane protein, which may serve as a transporter of polysaccharide to the bacterial cell surface. Whereas outer membrane proteins usually display an amphipathic beta-barrel fold, we show that PelC requires a C-terminal amphipathic alpha-helix for outer membrane insertion and function. Such a structural feature has only previously been reported for the Wza outer membrane protein of Escherichia coli, and our data suggest that this characteristic may be found in a large family of proteins, particularly outer membrane proteins specialized in polysaccharide transport.

PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport.

Pseudomonas aeruginosa is a gram-negative bacterium, opportunistic pathogen, which causes severe acute or chronic infections, as is the case with cystic fibrosis patients. Chronic infections are frequently accompanied by the development of the bacterial population into a specialized community called biofilm. The pelA-G gene cluster of P. aeruginosa has been shown to be involved in pellicle production and biofilm formation. The pel genes have been proposed to contribute to the formation of the exopolysaccharide-containing pellicle. However, the function and the subcellular localization of the seven different Pel proteins are poorly understood. Based on bioinformatics analysis, we have previously considered that PelF is a putative glycosyltransferase (GT4 family), whereas PelG is a Wzx-like polysaccharide transporter from the PST family. In this study we have further characterized the PelC protein. We have shown that PelC is an outer membrane lipoprotein. The N-terminal signal peptide of the PelC lipoprotein is sufficient to target the protein into the membranes. However, by constructing various PelC hybrid proteins we also proposed that efficient and functional outer membrane insertion of PelC requires not only the signal peptide and the lipid modification, but also requires the C-terminal domain of PelC. Because the gene encoding the outer membrane lipoprotein PelC is part of a putative gene cluster involved in exopolysaccharide biogenesis, we suggest that PelC is a new member of the outer membrane auxiliary (OMA) family of lipoprotein whose Wza, involved in Escherichia coli capsular polysaccharide transport, is an archetype.

Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes.

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.

The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with nosocomial infections and cystic fibrosis. Chronic bacterial infections are increasingly associated with the biofilm lifestyle in which microcolonies are embedded in an extracellular matrix. Screening procedures for identifying biofilm-deficient strains have allowed the characterization of several key determinants involved in this process. Biofilm-deficient P. aeruginosa PAK strains affected in a seven-gene cluster called pel were characterized. The pel genes encode proteins with similarity to components involved in polysaccharide biogenesis, of which PelF is a putative glycosyltransferase. PelG was also identified as a putative component of the polysaccharide transporter (PST) family. The pel genes were previously identified in the P. aeruginosa PA14 strain as required for the production of a glucose-rich matrix material involved in the formation of a thick pellicle and resistant biofilm. However, in PA14, the pel mutants have no clear phenotype in the initiation phase of attachment. It was shown that pel mutations in the PAK strain had little influence on biofilm initiation but, as in PA14, appeared to generate the least robust and mature biofilms. Strikingly, by constructing pel mutants in a non-piliated P. aeruginosa PAK strain, an unexpected effect of the pel mutation in the early phase of biofilm formation was discovered, since it was observed that these mutants were severely defective in the attachment process on solid surfaces. The pel gene cluster is conserved in other Gram-negative bacteria, and mutation in a Ralstonia solanacearum pelG homologue, ragG, led to an adherence defect.