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Control of bacterial phenotype and chromosomal gene expression by single plasmids of Lactococcus lactis IL594

ABSTRACT: Plasmid-free Lactococcus lactis IL1403 is one of the best-characterized representatives of lactic acid bacteria (LAB), intensively used in broad microbiology worldwide. Its parent strain, L. lactis IL594, contains seven plasmids (pIL1-pIL7) with resolved DNA sequences and an indicated role for overall plasmid load in enhancing host adaptive potential. To determine how individual plasmids manipulate the expression of phenotypes and chromosomal genes, we conducted global comparative phenotypic analyses combined with transcriptomic studies in plasmid-free L. lactis IL1403, multi-plasmid L. lactis IL594 and its single-plasmid derivatives. The presence of pIL2, pIL4 and pIL5 led to the most pronounced phenotypic differences in the metabolism of several carbon sources, including some β-glycosides and organic acids. The pIL5 plasmid also contributed to increased tolerance to some antimicrobial compounds and heavy metal ions, especially those in the toxic cation group. Comparative transcriptomics showed significant variation in the expression levels of up to 189 chromosomal genes due to the presence of single plasmids, and 435 unique chromosomal genes that are resultant of the activity of all plasmids, which may suggest that the observed phenotypic changes are not only the result of direct action of their own genes, but also originate from indirect actions through cross-talk between plasmids and the chromosome. The data obtained here indicate that plasmid maintenance leads to the development of important mechanisms of global gene regulation that provide changes in the central metabolic pathways and adaptive properties of L. lactis, and suggest the possibility of a similar phenomenon among other groups of bacteria.

ORGANISM(S): Lactococcus lactis

PROVIDER: GSE229414 | GEO | 2023/04/16

REPOSITORIES: GEO

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Project description:Bacteriophage infection of Lactococcus lactis strains used in the manufacture of fermented milk products is a major threat for the dairy industry. A greater understanding of the global molecular response of the bacterial host following phage infection has the potential to identify new targets for the design of phage control measures for biotechnological processes. In this study, we have used whole-genome oligonucleotide microarrays to gain insights into the genomic intelligence driving the instinctive response of L. lactis subsp. lactis IL1403 to the onset of a challenge with the lytic prolate-headed phage c2. Following phage adsorption, the bacterium differentially regulated the expression of 61 genes belonging to 14 functional categories, and mostly to cell envelope (12 genes), regulatory functions (11 genes), and carbohydrate metabolism (7 genes). The nature of the differentially regulated genes suggests the orchestration of a complex response involving induction of cell envelope stress proteins, D-alanylation of cell-wall lipoteichoic acids (LTAs), restoration of the proton motive force (PMF), and energy conservation. Increased D-alanylation of LTAs would act as an adsorption blocking mechanism, which we speculate may allow the survival of a small percent of the cell population when facing more realistic in vivo low titer-phage attacks. The modification of LTAs decoration in response to phage c2 adsorption also suggests these cell wall structures as possible primary receptors for this phage. Restoration of a physiological PMF is achieved by regulating the expression of genes affecting the two main components of the PMF, and serves to reverse a drastic depolarization of the host membrane caused by phage adsorption. Down-regulation of energy-consuming metabolic activities and a switch to anaerobic respiration helps the bacterium to save energy in order to sustain the PMF and the overall response to phage. We finally propose that the overall transcriptional response of L. lactis IL1403 to the phage stimuli is orchestrated by the concerted action of Phage Shock Proteins and of the bivalent transcriptional regulator SpxB following activation by the two-component system CesSR. To our knowledge, this represents the first detailed description in L. lactis, and probably in Gram-positive bacteria, of the molecular mechanisms involved in the host response to the membrane perturbation mediated by phage adsorption. Two-condition experiment: IL1403 vs. Bacteriophage c2-infected IL1403 cells. Biological replicates: 2 controls, 2 infected, independently grown and harvested. Two technical replicates per array.

Project description:Background: Lactococcus garvieae is a bacterial pathogen that affects different animal species and human. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study the genomic content of L. garvieae CECT 4531 was analyzed by bioinformatic tools and microarray-based comparative genomic hybridizations (CGH) experiments, using Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 as reference microorganisms. Results: The combination and integration of in silico analyses and in vitro (CGH) experiments performed between the reference microorganisms allowed establishing an inter-species hybridization framework with a detection threshold based on the sequence similarity ?70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of these genes are related to ribosomal, sugar metabolism or energy conversion systems. Some identified genes could be involved in the pathogenesis of L. garvieae infections. Conclusions: In this study a comparative analysis based on microarray interspecies hybridization and the use of bioinformatic tools were used for the first time to study the genetic content of L. garvieae CECT 4531. Towards this approach, we identified 267 potentially present genes in L. garvieae CECT 4531, some of which could be involved in the pathogenesis of L. garvieae infections, such as als or mycA. These results provide the first insight into the genome content of L. garvieae. Array-based comparative genome hybridization (CGH): The L. lactis subsp. lactis IL1403 and S. pneumoniae TIGR4 microarrays used for the CGH analysis were purchased from Eurogentec (Serain, Belgium). The L. lactis microarray contains 4608 spots: 2126 duplicated ORFs, 32 negative controls and 324 empty spots. The S. pneumoniae microarray contains 4608 spots: 2087 duplicated ORFs, 224 negative controls and 210 empty spots. The CGH experiments were performed by means of competitive hybridizations using DNA of L. lactis subsp. lactis IL1403 or S. pneumoniae TIGR4, depending on the array as positive controls. DNAs to be hybridized on the same array were labelled with Cy3-dUTP and Cy5-dUTP, respectively. For each microarray hybridization reaction, aliquots (1–2 µg) of labelled genomic DNAs of reference (labelled with Cy3) and test (labelled with Cy5) strains, were mixed in 45 µl EGT hybridization solution (Eurogentec, Serain, Belgium) and denatured at 65ºC for 2 min. The hybridization mixture was then loaded onto a microarray slide, covered with a coverslip and incubated at 38ºC overnight. Following hybridization, the slides were washed in 2 X SSC, 0.5% SDS for 5 min followed by 5 min in 1 X SSC, 0.25% SDS. Finally, slides were rinsed in 0.2 X SSC and dried by centrifugation. The results presented herein represent a compilation of sixteen separate CGH experiments: L. lactis subsp. lactis IL1403 arrays (reference microorganism) were hybridized with S. pneumoniae TIGR4 (test microorganism) (n=2); S. pneumoniae TIGR4 arrays (reference microorganism) were hybridized with L. lactis subsp. lactis IL1403 (test microorganism) (n=2); L. lactis subsp. lactis IL1403 arrays (reference microorganism) were hybridized with L.garvieae CECT 4531 (test microorganism) (n=8); S. pneumoniae TIGR4 arrays (reference microorganism) were hybridized with L.garvieae CECT 4531 (test microorganism) (n=4). Data acquisition and analysis: The microarray was scanned after hybridization using a Scanarray HT microarray scanner (Perkin-Elmer). The signal intensity of the two fluors was determined using ImaGene software (BioDiscovery, El Segundo, CA, USA). Microarray data were analysed using ImaGene software, Microsoft Excel and an in-house designed and built Microsoft Access database [30]. Gene calling was based on a signal-to-noise ratio (SNR) > 3 for each spot. After CGH experiments, a gene was considered to show a positive result when it was present in at least three of the four CGH assays. In the case of the L. garvieae CECT 4531 hybridizations with L. lactis subsp. lactis IL1403 arrays, it was necessary to perform a greater number of assays (n=8) due to the poor quality of one of the array batches used. Thus, the criteria chosen to determine a positive result in this case was when the gene was present in at least five of the eight CGH assays.

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Control of bacterial phenotype and chromosomal gene expression by single plasmids of Lactococcus lactis IL594

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