High-Resolution Chrono-Transcriptome of Lactococcus lactis Reveals That It Expresses Proteins with Adapted Size and pI upon Acidification and Nutrient Starvation.

Whole-genome transcriptional analyses performed on microorganisms are traditionally based on a small number of samples. To map transient expression variations, and thoroughly characterize gene expression throughout the growth curve of the widely used model organism Lactococcus lactis MG1363, gene expression data were collected with unprecedented time resolution. The resulting gene expression patterns were globally analyzed in several different ways to demonstrate the richness of the data and the ease with which novel phenomena can be discovered. When the culture moves from one growth phase to another, gene expression patterns change to such an extent that we suggest that those patterns can be used to unequivocally distinguish growth phases from each other. Also, within the classically defined growth phases, subgrowth phases were distinguishable with a distinct expression signature. Apart from the global expression pattern shifts seen throughout the growth curve, several cases of short-lived transient gene expression patterns were clearly observed. These could help explain the gene expression variations frequently observed in biological replicates. A method was devised to estimate a measure of unnormalized/absolute gene expression levels and used to determine how global transcription patterns are influenced by nutrient starvation or acidification of the medium. Notably, we inferred that L. lactis MG1363 produces proteins with on average lower pIs and lower molecular weights as the medium acidifies and nutrients get scarcer. IMPORTANCE This data set is a rich resource for microbiologists interested in common mechanisms of gene expression, regulation and in particular the physiology of L. lactis. Thus, similar to the common use of genome sequence data by the scientific community, the data set constitutes an extensive data repository for mining and an opportunity for bioinformaticians to develop novel tools for in-depth analysis.

pSEUDO, a genetic integration standard for Lactococcus lactis.

Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed.

Efficient overproduction of membrane proteins in Lactococcus lactis requires the cell envelope stress sensor/regulator couple CesSR.

BACKGROUND: Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor their folding state makes it difficult to foresee the yields at which one can obtain them or to predict which would be the optimal production host for a given protein. METHODS AND FINDINGS: We describe a rational design approach to improve the lactic acid bacterium Lactococcus lactis as a producer of membrane proteins. Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Growth of the BcaP-producing L. lactis strain and its capability to produce membrane proteins are severely hampered when the CesSR system itself or particular members of the CesSR regulon are knocked out, notably the genes ftsH, oxaA2, llmg_2163 and rmaB. Overexpressing cesSR reduced the growth defect, thus directly improving the production yield of BcaP. Applying this rationale to eukaryotic proteins, some of which are notoriously more difficult to produce, such as the medically-important presenilin complex, we were able to significantly diminish the growth defect seen in the wild-type strain and improve the production yield of the presenilin variant PS1Δ9-H6 more than 4-fold. CONCLUSIONS: The results shed light into a key, and perhaps central, membrane protein quality control mechanism in L. lactis. Modulating the expression of CesSR benefited the production yields of membrane proteins from different origins. These findings reinforce L. lactis as a legitimate alternative host for the production of membrane proteins.

The response of Lactococcus lactis to membrane protein production.

BACKGROUND: The biogenesis of membrane proteins is more complex than that of water-soluble proteins, and recombinant expression of membrane proteins in functional form and in amounts high enough for structural and functional studies is often problematic. To better engineer cells towards efficient protein production, we set out to understand and compare the cellular consequences of the overproduction of both classes of proteins in Lactococcus lactis, employing a combined proteomics and transcriptomics approach. METHODOLOGY AND FINDINGS: Highly overproduced and poorly expressed membrane proteins both resulted in severe growth defects, whereas amplified levels of a soluble substrate receptor had no effect. In addition, membrane protein overproduction evoked a general stress response (upregulation of various chaperones and proteases), which is probably due to accumulation of misfolded protein. Notably, upon the expression of membrane proteins a cell envelope stress response, controlled by the two-component regulatory CesSR system, was observed. CONCLUSIONS: The physiological response of L. lactis to the overproduction of several membrane proteins was determined and compared to that of a soluble protein, thus offering better understanding of the bottlenecks related to membrane protein production and valuable knowledge for subsequent strain engineering.

Amino acid accumulation limits the overexpression of proteins in Lactococcus lactis.

BACKGROUND: Understanding the biogenesis pathways for the functional expression of recombinant proteins, in particular membrane proteins and complex multidomain assemblies, is a fundamental issue in cell biology and of high importance for future progress in structural genomics. In this study, we employed a proteomic approach to understand the difference in expression levels for various multidomain membrane proteins in L. lactis cells grown in complex and synthetic media. METHODOLOGY/PRINCIPAL FINDINGS: The proteomic profiles of cells growing in media in which the proteins were expressed to high or low levels suggested a limitation in the availability of branched-chain amino acids, more specifically a too limited capacity to accumulate these nutrients. By supplying the cells with an alternative path for accumulation of Ile, Leu and/or Val, i.e., a medium supplement of the appropriate dipeptides, or by engineering the transport capacity for branched-chain amino acids, the expression levels could be increased several fold. CONCLUSIONS: We show that the availability of branched chain amino acids is a critical factor for the (over)expression of proteins in L. lactis. The forward engineering of cells for functional protein production required fine-tuning of co-expression of the branched chain amino acid transporter.

Contribution of the CesR-regulated genes llmg0169 and llmg2164-2163 to Lactococcus lactis fitness.

Lactococcus lactis is one of the main components of the starter cultures used in cheese manufacture. As starter, L. lactis must tolerate harsh conditions encountered either during their production in bulk quantities or during dairy products processing. To face these hostile conditions, bacteria monitor the environment and respond by modifying gene expression appropriately. Previous transcriptomic studies showed that the two component system CesSR is the main pathway that triggers the cell envelope stress response in L. lactis treated with lactococcin 972 (Lcn972), a cell wall synthesis inhibiting bacteriocin. Among the CesR-regulated genes, llmg0169 and the operon llmg2164-2163, encoding proteins of unknown function, are among the highest up-regulated genes after activation of CesSR. In this study, we have assessed the contribution of these genes to the survival of L. lactis to different technologically-relevant stresses. Overexpressing and knock-out mutants of the genes were generated and their viability to low pH, heat, freeze-drying, presence of NaCl, cell wall antimicrobials and lytic phages attack was compared to the wild type strain. The genes llmg0169 and llmg2164-2163 contributed differently to L. lactis fitness. L. lactis Deltallmg0169 was very sensitive to heat treatment while L. lactis Deltallmg2164 was more sensitive to NaCl. Absence of both genes also compromised viability at low pH. On the contrary, higher expression levels of llmg0169 and llmg2164-2163, up to 26- and 14-fold increase determined by qRT-PCR, respectively, did not enhance L. lactis survival in any of the above stressful conditions (heat, pH and NaCl) or after freeze-drying. All the mutants displayed a similar phage susceptibility profile. Overexpression of llmg2164-2163 seemed to specifically protect L. lactis against the bacteriocin Lcn972 but not against other cell wall active antimicrobials. Based on our phenotypic analysis, the investigated genes are required to mount a proper response to guarantee survival of L. lactis under technologically-relevant stresses and their functionality could be a useful marker to select robust dairy starters.