Combining Modules for Versatile and Optimal Labeling of Lactic Acid Bacteria: Two pMV158-Family Promiscuous Replicons, a Pneumococcal System for Constitutive or Inducible Gene Expression, and Two Fluorescent Proteins

Abstract

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis , we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter P X or P M from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying P M also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8–45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.

Acidic pH Decreases the Endonuclease Activity of Initiator RepB and Increases the Stability of the Covalent RepB-DNA Intermediate while Has Only a Limited Effect on the Replication of Plasmid pMV158 in Lactococcus lactis

“…Lactococcal gDNA, used as template for real-time quantitative PCR (qPCR), was isolated as described in ( Garay-Novillo et al, 2019 ). Purified gDNA was digested with EcoRI, which linearizes the pMV158 plasmid DNA but leaves intact the template DNA segments containing the plasmidic and chromosomal amplicons that will be amplified in the qPCR assays.…”

“…For the determination of the PCN per chromosomal equivalent by qPCR, two primer pair sets specific to the PcrA helicase single-copy reference gene ( pcrA ) of L. lactis MG1363 ( Wegmann et al, 2007 ) and to the RepB replication protein gene ( repB ) of pMV158 were used ( Garay-Novillo et al, 2019 ). Criteria used during primer design were that primers had a predicted Tm of ∼59°C and that they generated amplicons of ∼140 bp in length.…”

“…In each trial, triplicate samples of the three different amounts of template were analyzed. PCN of pMV158 at the different pH conditions studied was calculated as described ( Garay-Novillo et al, 2019 ).…”

Mutations Selected After Exposure to Bacteriocin Lcn972 Activate a Bce-Like Bacitracin Resistance Module in Lactococcus lactis

“…After induction with bacitracin (1 and 5 μg/ml) or Lcn972 (80, 40, and 20 AU/ml), cells (1 ml) were washed with saline phosphate buffer (PBS), pH 7.3, and collected in half of the initial volume. Cell suspensions were kept in the dark for 3 h for mCherry maturation (Garay-Novillo et al, 2019). Fluorescence (F) of 20-μl aliquots was quantified in a 7500 Fast Real-Time PCR System (Applied Biosystems, Warrington, UK) using the built-in presence/absence protocol for detecting ROX, a fluorophore with similar excitation (Ex.…”

“French Phage Network” Annual Conference—Fifth Meeting Report

“…Lactic acid bacteria (LAB) as Streptococcus thermophilus are widely used for industrial food fermentation, but also generate great interest for their food-grade expression capabilities (proteins or metabolites) [15,16]. To do so, replicating plasmids are often used to introduce genetic material into LAB cells [17,18], but these plasmids need to be maintained into the cells. This can be achieved by several methods, including recently the use of CRISPR-Cas technology [19,20].…”