RESUMO
Bacterial DNA methyltransferases (MTases) function in restriction modification systems, cell cycle control, and the regulation of gene expression. DnmA is a recently described DNA MTase that forms N6-methyladenosine at nonpalindromic 5'-GACGAG-3' sites in Bacillus subtilis, yet how DnmA activity is regulated is unknown. To address DnmA regulation, we tested substrate binding in vitro and found that DnmA binds poorly to methylated DNA and to an RNA-DNA hybrid with the DNA recognition sequence. Further, DnmA variants with amino acid substitutions that disrupt cognate sequence recognition or catalysis also bind poorly to DNA. Using superresolution fluorescence microscopy and single-molecule tracking of DnmA-PAmCherry, we characterized the subcellular DnmA diffusion and detected its preferential localization to the replisome region and the nucleoid. Under conditions where the chromosome is highly methylated, upon RNA-DNA hybrid accumulation, or with a DnmA variant with severely limited DNA binding activity, DnmA is excluded from the nucleoid, demonstrating that prior methylation or accumulation of RNA-DNA hybrids regulates the association of DnmA with the chromosome in vivo. Furthermore, despite the high percentage of methylated recognition sites and the proximity to putative endonuclease genes conserved across bacterial species, we find that DnmA fails to protect B. subtilis against phage predation, suggesting that DnmA is functionally an orphan MTase involved in regulating gene expression. Our work explores the regulation of a bacterial DNA MTase and identifies prior methylation and RNA-DNA hybrids as regulators of MTase localization. These MTase regulatory features could be common across biology. IMPORTANCE DNA methyltransferases (MTases) influence gene expression, cell cycle control, and host defense through DNA modification. Predicted MTases are pervasive across bacterial genomes, but the vast majority remain uncharacterized. Here, we show that in the soil microorganism Bacillus subtilis, the DNA MTase dnmA and neighboring genes are remnants of a phage defense system that no longer protects against phage predation. This result suggests that portions of the bacterial methylome may originate from inactive restriction modification systems that have maintained methylation activity. Analysis of DnmA movement in vivo shows that active DnmA localizes in the nucleoid, suggesting that DnmA can search for recognition sequences throughout the nucleoid region with some preference for the replisome. Our results further show that prior DNA methylation and RNA-DNA hybrids regulate DnmA dynamics and nucleoid localization, providing new insight into how DNA methylation is coordinated within the cellular environment.
Assuntos
Bacteriófagos , Metiltransferases , Metiltransferases/genética , Metiltransferases/metabolismo , Metilação de DNA , RNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Bacteriófagos/genéticaRESUMO
Acidification and nutrient depletion by dairy starter cultures is often sufficient to prevent outgrowth of pathogens during post-processing of cultured dairy products. In the case of cottage cheese, however, the addition of cream dressing to the curd and subsequent cooling procedures can create environments that may be hospitable for the growth of Listeria monocytogenes. We report on a non-bacterio-cinogenic Lacticaseibacillus rhamnosus strain that severely limits the growth potential of L. monocytogenes in creamed cottage cheese. The main mechanism underlying Listeria spp. inhibition was found to be caused by depletion of manganese (Mn), thus through competitive exclusion of a trace element essential for the growth of many microorganisms. Growth of Streptococcus thermophilus and Lactococcus lactis that constitute the starter culture, on the other hand, were not influenced by reduced Mn levels. Addition of L. rhamnosus with Mn-based bioprotective properties during cottage cheese production therefore offers a solution to inhibit undesired bacteria in a bacteriocin-independent fashion.
RESUMO
Manganese (Mn2+) is an essential trace element within organisms spanning the entire tree of life. In this review, we provide an overview of Mn2+ transport and the regulation of its homeostasis in bacteria, with a focus on its functions beyond being a cofactor for enzymes. Crucial differences in Mn2+ homeostasis exist between bacterial species that can be characterized to have an iron- or manganese-centric metabolism. Highly iron-centric species require minimal Mn2+ and mostly use it as a mechanism to cope with oxidative stress. As a consequence, tight regulation of Mn2+ uptake is required, while organisms that use both Fe2+ and Mn2+ need other layers of regulation for maintaining homeostasis. We will focus in detail on manganese-centric bacterial species, in particular lactobacilli, that require little to no Fe2+ and use Mn2+ for a wider variety of functions. These organisms can accumulate extraordinarily high amounts of Mn2+ intracellularly, enabling the nonenzymatic use of Mn2+ for decomposition of reactive oxygen species while simultaneously functioning as a mechanism of competitive exclusion. We further discuss how Mn2+ accumulation can provide both beneficial and pathogenic bacteria with advantages in thriving in their niches.
Assuntos
Ferro , Manganês , Bactérias , Transporte Biológico , Estresse OxidativoRESUMO
Lactococcus lactis cheese starter cultures typically contain a mix of many strains and may include variants that produce and/or tolerate the antimicrobial bacteriocin nisin. Nisin is well-established as an effective agent against several undesirable Gram-positive bacteria in cheese and various other foods. In the current study, we have examined the effect of nisin on 710 individual L. lactis strains during milk fermentations. Changes in milk acidification profiles with and without nisin exposure, ranging from unaltered acidification to loss of acidification, could be largely explained by the type(s) and variants of nisin immunity and nisin degradation genes present, but surprisingly, also by genotypic lineage (L. lactis ssp. cremoris vs. ssp. lactis). Importantly, we identify that nisin degradation by NSR is frequent among L. lactis and therefore likely the main mechanism by which dairy-associated L. lactis strains tolerate nisin. Insights from this study on the strain-specific effect of nisin tolerance and degradation during milk acidification is expected to aid in the design of nisin-compatible cheese starter cultures.
RESUMO
The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.
Assuntos
Adenosina/análogos & derivados , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Adenosina/análise , Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , Metilação de DNA , Enzimas de Restrição-Modificação do DNA , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism. Among many other things, this has provided an unprecedented view of the major gene regulatory pathways involved in nitrogen and carbon metabolism and their overlap, and has led to the blossoming of the field of L. lactis systems biology. All of these advances have made L. lactis the paradigm of the LAB. This review will deal with the exciting path along which the research on the genetics of and gene regulation in L. lactis has trodden.
Assuntos
Produtos Fermentados do Leite/microbiologia , Regulação Bacteriana da Expressão Gênica/genética , Engenharia Genética/métodos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Fermentação/genética , Técnicas de Transferência de Genes , Genoma Bacteriano/genética , Plasmídeos/genéticaRESUMO
To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998) and Femino et al. (1998). Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow. Here, an overview will be given of fluorescent techniques that can be used to reveal specific RNA molecules inside fixed and living single bacterial cells. It includes a critical evaluation of their caveats as well as potential solutions.
RESUMO
By using fluorescence imaging, we provide a time-resolved single-cell view on coupled defects in transcription, translation, and growth during expression of heterologous membrane proteins in Lactococcus lactis. Transcripts encoding poorly produced membrane proteins accumulate in mRNA-dense bodies at the cell poles, whereas transcripts of a well-expressed homologous membrane protein show membrane-proximal localization in a translation-dependent fashion. The presence of the aberrant polar mRNA foci correlates with cessation of cell division, which is restored once these bodies are cleared. In addition, activation of the heat-shock response and a loss of nucleoid-occluded ribosomes are observed. We show that the presence of a native-like N-terminal domain is key to SRP-dependent membrane localization and successful production of membrane proteins. The work presented gives new insights and detailed understanding of aberrant membrane protein biogenesis, which can be used for strategies to optimize membrane protein production.