Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the omega subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc(2)155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting.     

Mutual effects of MinD-membrane interaction: II. Domain structure of the membrane enhances MinD binding

MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on pre-existing anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.     

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis> Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.     

Cell Death is Associated with Reduced Base Excision Repair During Chronic Alcohol Administration in Adult Rat Brain

The cell death cascades in different brain regions namely hippocampus and frontal cortex of rats fed with 10% (v/v) ethanol for 12 weeks, was examined. After Western blotting, different cell death associated proteins displayed differential activation in the two regions observed. In hippocampus, activated caspase-3 and caspase-7 resulted in subsequent cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). Cytochrome c release to cytosol and apoptosis inducing factor (AIF) translocation to nucleus was marginal. B-cell leukemia/lymphoma-2 (Bcl-2) translocation to cytosol was significant whereas Bcl-2-associated X protein (Bax) and Bcl-associated death protein (Bad) were largely located in cytosol. Further, upregulation of N-methyl d-aspartate receptor subunit 1 (NMDAR1), N-methyl d-aspartate receptor subunit 2B (NMDAR2B), N-methyl d-aspartate receptor subunit 2C (NMDAR2C) and activation of calpains were observed. In frontal cortex, caspase-3 activation, cleavage of PARP-1 and nuclear translocation of AIF were more pronounced. Moreover, cytochrome c release to cytosol, Bcl-2 translocation to cytosol was evident. However, levels of Bax, Bad, NMDA receptor subunits, and calpains were unaffected. Apoptosis was further substantiated by in situ staining for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). Results of the current study revealed that frontal cortex exhibits a higher level of ethanol-induced apoptosis relative to hippocampus. DNA polymerase beta assay and immunoblot showed significant loss in base excision repair in ethanol treated group.     

Radiobiology of alpha particles. III. Cell inactivation by alpha-particle traversals of the cell nucleus

Cell inactivation after exposure to collimated 3.5-MeV alpha particles in three hamster cell lines, V79, CHO-10B, and HS-23, one mouse cell line, C3H 10T1/2, and a human skin fibroblast cell line were studied. Several parameters were investigated for each cell line. Theoretical calculations were performed to find the distribution of energy deposited in the nuclear volume for each cell line. The mean number of alpha-particle traversals required to induce a lethal lesion varied between two for HS-23 cells and six for C3H 10T1/2 cells. The number of traversals per unit area and the total track length of alpha particles that inactivated a cell were found to be nearly constant for the hamster and mouse cell lines. These quantities were found to be lower for the human skin fibroblast cell line. The RBE values for all cell lines were found to be about 3.8 at 10% survival. Thus cell lines that are more sensitive to alpha radiation are also more sensitive to gamma radiation. The average number of alpha-particle traversals producing a single lethal lesion is greater than one. The passages of alpha particles through the cell nucleus that do not kill the cell may lead to carcinogenic effects.     

Pheromone trail following in the ant Lasius niger: high accuracy and variability but no effect of task state

The use of pheromone trails in ant colony organization is an important model for understanding collective decision‐making and complex adaptive systems. The ant Lasius niger L. (Hymenoptera: Fomicidae) is one of the main model organisms used for such studies. Key to understanding pheromone trail use by ants is knowing how well trails are followed. The results of a previous study suggest that L. niger trail following is poor, with between 60% and 70% accuracy at a T bifurcation. It is hypothesized that the true trail following accuracy is higher, and that the low accuracy reported previously is the result of a methodological error. Specifically, it is hypothesized that ‘task state’ (i.e. what the ants ‘thought they were doing’) affected pheromone following accuracy. In the present study, the task state of the ants is set experimentally to one of three states: scouting (completely naive), recruited (having information that food has been found, but not where it is) and shuttling (having a strong memory of the location of a food source). Trail following accuracy is tested for each group. Trail following is found to be more accurate than previously reported: 83%, 82% and 74% correct decisions for scouts, recruits and shuttlers, respectively. However, the difference between the three groups is not significant. Importantly, very high inter‐trial variation is reported both in the present study and in experiments from other research groups. This variation is unexplainable by trail strengths or colony‐level differences, and is highlighted as an important factor when experimentally measuring trail following.     

Synthesis and evaluation of 3″- and 4″-deoxy and -fluoro analogs of the immunostimulatory glycolipid,KRN7000

Four 3″- and 4″-deoxy and -fluorogalactosyl ceramides were synthesized, and their ability to stimulate iNKT cells, based on levels of IL-2 production, was assessed in three NKT cell receptor hybridomas. In two of the hybridomas, 1.2 and 2H4, all of the analogs were immunostimulatory, while in the 1.4 hybridoma only the 4″-fluoro analog led to the production of significant levels of IL-2.