Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P_M promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P_M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P_M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Molecular analysis of the malR gene of Clostridium butyricum NCIMB 7423, a member of the LacI-GalR family of repressor proteins

Deletion of a region of DNA 5′ to a previously characterised malQ gene of Clostridium butyricum resulted in increased production of the enzyme activity encoded by malQ, 4-α-glucanotransferase. Nucleotide sequence analysis revealed the presence of an open reading frame capable of encoding a protein of 335 amino acids. This protein was found to share 33% amino acid sequence identity with the Bacillus subtilis CcpA (catabolite control protein) repressor, 28% identity with the Streptomyces coelicolor MalR repressor, and 30%, 25%, and 21% amino acid identity with the Escherichia coli repressors GalR, LacI and MalI, respectively. The amino-terminal domain was predicted to be able to form a helix-turn-helix structure, and shared highest similarity with the equivalent functional domain from the E. coli LacI repressor. Interruption of malR by the generation of a frameshift mutation led to a 10-fold increase in MalQ activity. These data suggest that the identified open reading frame encodes a repressor of the C. butyricum malQ gene, and of the adjacent malP gene. The gene has, therefore, been designated malR, and its encoded gene product MalR.     

RNA-seq analysis of green tea polyphenols modulation of differently expressed genes in Enterococcus faecalis under low pH

Enterococcus faecasslis (E. faecalis) is a resident bacterium in the host. The increase in internal stress like low pH may affect the biological effects of E. faecalis. The prebiotic-like function of tea polyphenols can enhance the beneficial effects of its tolerance to environmental stress. In this study, RNA-sequence analysis was used to explore the protective effect of green tea polyphenols (GTP) on E. faecalis under low pH stress. A total of 28 genes were found to be responsive to GTP under low pH stress, including 16 up-regulated and 12 down-regulated. GTP intervention can partly relieve some undesired negative influences, such as the down-regulation of the base excision repair gene and amino acid transport and metabolism gene. The significantly changes were associated with selenocompound metabolism and aminoacyl-tRNA biosynthesis after the intervention of GTP. The present study provided new insights into the growth and continuous adaptation of E. faecalis under stress.     

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6′‐phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.     

Expression of the Agmatine Deiminase Pathway in Enterococcus faecalis Is Activated by the AguR Regulator and Repressed by CcpA and PTSMan Systems

Although the agmatine deiminase system (AgDI) has been investigated in Enterococcus faecalis, little information is available with respect to its gene regulation. In this study we demonstrate that the presence of exogenous agmatine induces the expression of agu genes in this bacterium. In contrast to the homologous and extensively characterized AgDI system of S. mutants, the aguBDAC operon in E. faecalis is not induced in response to low pH. In spite of this, agmatine catabolism in this bacterium contributes by neutralizing the external medium while enhancing bacterial growth. Our results indicate that carbon catabolic repression (CCR) operates on the AgDI system via a mechanism that involves interaction of CcpA and P-Ser-HPr with a cre site found in an unusual position considering the aguB promoter (55 nt upstream the +1 position). In addition, we found that components of the mannose phosphotransferase (PTS^Man) system also contributed to CCR in E. faecalis since a complete relief of the PTS-sugars repressive effect was observed only in a PTS^Man and CcpA double defective strain. Our gene context analysis revealed that aguR is present in oral and gastrointestinal microorganisms. Thus, regulation of the aguBDAC operon in E. faecalis seems to have evolved to obtain energy and resist low pH conditions in order to persist and colonize gastrointestinal niches.     

The role of amylomaltase in maltose metabolism in the cytosol of photosynthetic cells

Transitory starch is stored during the day inside chloroplasts and then broken down at night for export. Recent data indicate that maltose is the major form of carbon exported from the chloroplast at night but its fate in the cytosol is unknown. An amylomaltase gene (malQ) cloned from Escherichia coli is necessary for maltose metabolism in E. coli. We investigated whether there is an amylomaltase in the cytosol of plant leaves and the role of this enzyme in plants. Two mutants of Arabidopsis thaliana (L) Heynh. were identified in which the gene encoding a putative amylomaltase enzyme [disproportionating enzyme 2, DPE2 (DPE1 refers to the plastid version of this enzyme)] was disrupted by a T-DNA insertion. Both dpe2-1 and dpe2-2 plants exhibited a dwarf phenotype and accumulated a large amount of maltose. In addition, dpe2 mutants accumulated starch and a water-soluble, ethanol/KCl-insoluble maltodextrin in their chloroplasts. At night, the amount of sucrose in dpe2 plants was lower than that in wild-type plants. These results show that Arabidopsis has an amylomaltase that is involved in the conversion of maltose to sucrose in the cytosol. We hypothesize that knocking out amylomaltase blocks the conversion from maltose to sucrose, and that the higher amount of maltose feeds back to limit starch degradation reactions in chloroplasts. As a result, dpe2 plants have higher maltose, higher starch, and higher maltodextrin but lower nighttime sucrose than wild-type plants. Finally, we propose that maltose metabolism in the cytosol of Arabidopsis leaves is similar to that in the cytoplasm of E. coli.Abbreviations F6P fructose 6-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - GTase glucanotransferase     

The role of the CroR response regulator in resistance of Enterococcus faecalis to D‐cycloserine is defined using an inducible receiver domain

Enterococcus faecalis is an opportunistic multidrug‐resistant human pathogen causing severe nosocomial infections. Previous investigations revealed that the CroRS two‐component regulatory pathway likely displays a pleiotropic role in E. faecalis, involved in virulence, macrophage survival, oxidative stress response as well as antibiotic resistance. Therefore, CroRS represents an attractive potential new target for antibiotherapy. In this report, we further explored CroRS cellular functions by characterizing the CroR regulon: the ‘domain swapping’ method was applied and a CroR chimera protein was generated by fusing the receiver domain from NisR to the output domain from CroR. After demonstrating that the chimera CroR complements a croR gene deletion in E. faecalis (stress response, virulence), we conducted a global gene expression analysis using RNA‐Seq and identified 50 potential CroR targets involved in multiple cellular functions such as cell envelope homeostasis, substrate transport, cell metabolism, gene expression regulation, stress response, virulence and antibiotic resistance. For validation, CroR direct binding to several candidate targets was demonstrated by EMSA. Further, this work identified alr, the gene encoding the alanine racemase enzyme involved in E. faecalis resistance to D‐cycloserine, a promising antimicrobial drug to treat enterococcal infections, as a member of the CroR regulon.     

The opportunistic pathogen Enterococcus faecalis resists phagosome acidification and autophagy to promote intracellular survival in macrophages

While many strains of Enterococcus faecalis have been reported to be capable of surviving within macrophages for extended periods, the exact mechanisms involved are largely unknown. In this study, we found that after phagocytosis by macrophages, enterococci‐containing vacuoles resist acidification, and E. faecalis is resistant to low pH. Ultrastructural examination of the enterococci‐containing vacuole by transmission electron microscopy revealed a single membrane envelope, with no evidence of the classical double‐membraned autophagosomes. Western blot analysis further confirmed that E. faecalis could trigger inhibition of the production of LC3‐II during infection. By employing cells transfected with RFP‐LC3 plasmid and infected with GFP‐labelled E. faecalis, we also observed that E. faecalis was not delivered into autophagosomes during macrophage infection. While these observations indicated no role for autophagy in elimination of intracellular E. faecalis, enhanced production of reactive oxygen species and nitric oxide were keys to this process. Stimulation of autophagy suppressed the intracellular survival of E. faecalis in macrophages in vitro and decreased the burden of E. faecalis in vivo. In summary, the results from this study offer new insights into the interaction of E. faecalis with host cells and may provide a new approach to treatment of enterococcal infections.