Linear DNA plasmids of yeasts

Abstract Proteinaceous antimicrobial compounds are produced by a diversity of species ranging from bacteria to humans. This review focuses on the mode of action of pore-forming bacteriocins produced by Gram-positive bacteria. The mechanism of action of specific immunity proteins, which protect the producer strains from the lethal action of their own products (producer self-protection), are also discussed.     

S-cysteinylation is a general mechanism for thiol protection of Bacillus subtilis proteins after oxidative stress

S-Thiolation is crucial for protection and regulation of thiol-containing proteins during oxidative stress and is frequently achieved by the formation of mixed disulfides with glutathione. However, many Gram-positive bacteria including Bacillus subtilis lack the low molecular weight (LMW) thiol glutathione. Here we provide evidence that S-thiolation by the LMW thiol cysteine represents a general mechanism in B. subtilis. In vivo labeling of proteins with [(35)S]cysteine and nonreducing two-dimensional PAGE analyses revealed that a large subset of proteins previously identified as having redox-sensitive thiols are modified by cysteine in response to treatment with the thiol-specific oxidant diamide. By means of multidimensional shotgun proteomics, the sites of S-cysteinylation for six proteins could be identified, three of which are known to be S-glutathionylated in other organisms.     

Synthesis and in vitro antimicrobial studies of medicinally important novel N-alkyl and N-sulfonyl derivatives of 1-[bis(4-fluorophenyl)-methyl]piperazine

A series of novel substituted 1-[bis(4-fluorophenyl)-methyl]piperazine derivatives (4a-g) and (5h-m) have been synthesized. The synthesized compounds were characterized by IR and 1H NMR. All the synthesized compounds were evaluated in vitro for their efficacy as antimicrobial agents against representative strains of Gram-positive (Staphylococcus aureus ATCC 25953, Streptococcus pneumoniae ATCC 49619, Bacillus cereus 11778, and Bacillus subtilis 6051) and Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 2853, Proteus vulgaris ATCC 2853, and Salmonella typhi ATCC 9484) by paper disc diffusion and microdilution methods. Among the newly synthesized compounds 4e, 5l, and 5m showed potent antimicrobial activities, when compared to the standard drug.     

Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis

Bacterial alarmone (p)ppGpp, is a global regulator responsible for the stringent control. Two homologous (p)ppGpp synthetases, RelA and SpoT, have been identified and characterized in Escherichia coli, whereas Gram-positive bacteria such as Bacillus subtilis have been thought to possess only a single RelA-SpoT enzyme. We have now identified two genes, yjbM and ywaC, in B. subtilis that encode a novel type of alarmone synthetase. The predicted products of these genes are relatively small proteins ( approximately 25 kDa) that correspond to the (p)ppGpp synthetase domain of RelA-SpoT family members. A database survey revealed that genes homologous to yjbM and ywaC are conserved in certain bacteria belonging to Firmicutes or Actinobacteria phyla but not in other phyla such as Proteobacteria. We designated the proteins as small alarmone synthetases (SASs) to distinguish them from RelA-SpoT proteins. The (p)ppGpp synthetase function of YjbM and YwaC was confirmed by genetic complementation analysis and by in vitro assay of enzyme activity. Molecular genetic analysis also revealed that ywaC is induced by alkaline shock, resulting in the transient accumulation of ppGpp. The SAS proteins thus likely function in the biosynthesis of alarmone with a mode of action distinct from that of RelA-SpoT homologues.     

Synthesis, antimicrobial and anticancer activities of a novel series of diphenyl 1-(pyridin-3-yl)ethylphosphonates

A novel series of diphenyl 1-(arylamino)-1-(pyridin-3-yl)ethylphosphonates 1-5 was obtained in high yields from reactions of 3-acetyl pyridine with aromatic amines and triphenylphosphite in the presence of lithium perchlorate as a catalyst. The structures of the synthesized compounds were confirmed by IR, (1)H NMR spectral data and microanalyses. Compounds 1-5 showed high antimicrobial activities against Escherichia coli (NCIM2065) as a Gram-negative bacterium, Bacillus subtilis (PC1219) and Staphylococcus aureus (ATCC25292) as Gram-positive bacteria and Candidaalbicans and Saccharomyces cerevisiae as fungi, at low concentrations (10-100 μg/mL). Also, the synthesized compounds showed significant cytotoxicity anticancer activities against liver carcinoma cell line (HepG2) and human breast adenocarcinoma cell line (MCF7). The lethal dose of the synthesized compounds was also determined and indicated that most compounds are safe to use.     

Synthesis of novel pyrroles and fused pyrroles as antifungal and antibacterial agents

Pyrroles and its fused forms possess antimicrobial activities, they can easily interact with biomolecules of living systems. A series of substituted pyrroles, and its fused pyrimidines and triazines forms have been synthesised, all newly synthesised compound structures were confirmed by spectroscopic analysis. Generally, the compounds inhibited growth of some important human pathogens, the best effect was given by: 2a, 3c, 4d on Gram-positive bacteria and was higher on yeast (C. albicans), by 5c on Gram-negative bacteria and by 5a then 3c on filamentous fungi (A. fumigatus and F. oxysporum). Such results present good antibacterial and antifungal potential candidates to help overcome the global problem of antibiotic resistance and opportunistic infections outbreak. Compound 3c gave the best anti-phytopathogenic effect at a 50-fold lower concentration than Kocide 2000, introducing a safe commercial candidate for agricultural use. The effect of the compounds on DNA was monitored to detect the mode of action.     

Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria

Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci.     

Synthesis, structural characterization and antimicrobial activities of 4- and 6-coordinate nickel(II) complexes with three thiosemicarbazones and semicarbazone ligands

To investigate the relationship between antimicrobial activities and the molecular structures of nickel(II) complexes with thiosemicarbazone and semicarbazone ligands, nickel(II) complexes with ligands Hmtsc, Hatsc, Hasc and H2dmtsc, were prepared and characterized by elemental analysis, FT-IR, 1H and 13C NMR spectroscopies, magnetic susceptibility measurements, UV-Vis absorption spectra, TG/DTA and single-crystal X-ray analysis. Their antimicrobial activities were evaluated by the MIC against four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts (C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The 4-coordinate, diamagnetic nickel(II) complexes showed antimicrobial activities which were different from those of free ligands or the starting nickel(II) compounds; [Ni(mtsc)(OAc)] 1 showed selective and effective antimicrobial activities against two Gram-positive bacteria (B. subtilis and S. aureus) and modest activities against a yeast (S. cerevisiae), [Ni(mtsc)Cl] 3 exhibited moderate activities against a Gram-positive bacterium (S. aureus), and [Ni(atsc)(OAc)] 5 showed modest activities against two Gram-positive bacteria (B. subtilis and S. aureus). On the other hand, the 6-coordinate, paramagnetic nickel(II) complexes with two protonated or deprotonated ligands ([Ni(mtsc)2] 2, [Ni(atsc)(mtsc)] 4, [Ni(atsc)2] 6, [Ni(Hatsc)2](NO3)(2)7, [Ni(Hatsc)2]Cl(2)8 and [Ni(Hasc)2](OAc)(2)9) and the sterically crowded 4-coordinate, diamagnetic nickel(II) complex ([Ni(dmtsc)] 10) did not inhibit the growth of the test organisms. The structure-activity correlation in this series of nickel(II) complexes was discussed based on their ligand-replacement abilities.     

Two novel cytotoxic and antimicrobial triterpenoids from Pseudolarix kaempferi.

Two novel antimicrobial and cytotoxic triterpenoids, isopseudolarifuroic acids A (1) and B (2), were isolated from the bark of Pseudolarix kaempferi. The structural elucidation of two novel compounds was carried out mainly by spectroscopic methods, and also by computer modeling. Compounds 1 and 2 exhibited significant cytotoxic activities against several tumor cell lines. Compound 1 also showed most potent antimicrobial activities against both Gram-positive and Gram-negative bacteria.     

Design and synthesis of spiro[indole-thiazolidine]spiro[indole-pyrans] as antimicrobial agents

A series of novel spiro[indole-thiazolidine]spiro[indole-pyran] derivatives were synthesized from N-(bromoalkyl)indol-2,3-diones via monospiro-bisindole intermediates; the two indole nuclei being connected via N-(CH(2))(n)-N linker. Synthesized compounds were evaluated for their antimicrobial activities in vitro against three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermis), four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia) as well as four fungi (Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Candida albicans) using Cup plate method. Bis spiro-indoles exhibited stronger antibacterial and antifungal efficiency than their corresponding mono spiro-indoles. Compound 10e, the most active derivative was shown to inhibit the growth of all bacterial strains and two fungal strains (A. niger and C. albicans).     

Quorum sensing by peptide pheromones and two-component signal-transduction systems in Gram-positive bacteria

Cell-density-dependent gene expression appears to be widely spread in bacteria. This quorum-sensing phenomenon has been well established in Gram-negative bacteria, where N -acyl homoserine lactones are the diffusible communication molecules that modulate cell-density-dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell-density- or growth-phase-dependent manner in Gram-positive bacteria. Examples of such quorum-sensing modes in Gram-positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae , the virulence response in Staphylococcus aureus , and the production of antimicrobial peptides by several species of Gram-positive bacteria including lactic acid bacteria. Cell-density-dependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post-translationally processed peptide that is secreted by a dedicated ATP-binding-cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two-component signal-transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide-pheromone production.     

In Vitro Synergistic Activities of the Hybrid Antimicrobial Peptide MelitAP-27 in Combination with Conventional Antibiotics Against Planktonic and Biofilm Forming Bacteria

The extensive use of antibiotics for the treatment of human infections during the last few decades has led to a dramatic increase in the emergence of multidrug-resistant bacteria (MDRB) among various bacterial strains. Global research is currently focused on finding novel alternative agents with different mechanisms of action rather than the use of conventional antibiotics to counteract the threat of bacterial and biofilm infections. Antimicrobial peptides represent promising alternative agents for conventional antibiotics as these molecules display a broad spectrum of activity against several microorganisms. Recently, we have designed a novel hybrid antimicrobial peptide named MelitAP-27. This peptide has been found to display potent broad spectrum and selective in vitro antimicrobial activities against a wide range of Gram-positive and Gram-negative bacteria. In the present study, the in vitro antimicrobial and antibiofilm activities of the peptide alone and in combination with five different types of antibiotics were assessed against wild-type and resistant Gram-positive and Gram-negative bacterial strains. Our results showed that most of the combination groups displayed a synergistic mode of action against planktonic and biofilm forming bacteria which resulted in decreasing the effective MIC values for MelitAP-27 to the nanomolar concentrations. These effective concentrations were associated with negligible toxicities on mammalian cells. The results of our study indicate that combinations of MelitAP-27 with conventional antibiotics may be pursued as a potential novel treatment strategy against MDRB and biofilm forming bacteria.     

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.     

Histidine kinases as targets for new antimicrobial agents

The emergence and spread of hospital acquired multi drug resistant bacteria present a need for new antibiotics with innovative mode of action. Advances in molecular microbiology and genomics have led to the identification of numerous bacterial genes coding for proteins that could potentially serve as targets for antibacterial compounds. Histidine kinase promoted two-component systems are extremely common in bacteria and play an important role in essential signal transduction for adapting to bacterial stress. Since signal transduction in mammals occurs by a different mechanism, inhibition of histidine kinases could be a potential target for antimicrobial agents. This review will summarize our current knowledge of the structure and function of histidine kinase and the development of antibiotics with a new mode of action: targeting histidine kinase promoted signal transduction and its subsequent regulation of gene expression system.