A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity

Diadenosine polyphosphates, Ap((2-7))A, which contain two adenosines in a 5',5' linkage through phosphodiester bonds involving 2-7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap(6)A. Synthetic Ap(6)A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap(6)A in the CCS. Thus, the present work reveals a novel role for Ap(6)A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population

Asymmetric cell division in Mycobacterium tuberculosis and its unique features

Recently, several reports showed that about 80 % of mid-log phase Mycobacterium smegmatis, Mycobacterium marinum, and Mycobacterium bovis BCG cells divide symmetrically with 5-10 % deviation in the septum position from the median. However, the mode of cell division of the pathogenic mycobacterial species, Mycobacterium tuberculosis, remained unclear. Therefore, in the present study, using electron microscopy, fluorescence microscopy of septum- and nucleoid-stained live and fixed cells, and live cell time-lapse imaging, we show the occurrence of asymmetric cell division with unusually deviated septum/constriction in 20 % of the 15 % septating M. tuberculosis cells in the mid-log phase population. The remaining 80 % of the 15 % septating cells divided symmetrically but with 2-5 % deviation in the septum/constriction position, as reported for M. smegmatis, M. marinum, and M. bovis BCG cells. Both the long and the short portions of the asymmetrically dividing M. tuberculosis cells with unusually deviated septum contained nucleoids, thereby generating viable short and long cells from each asymmetric division. M. tuberculosis short cells were acid fast positive and, like the long cells, further readily underwent growth and division to generate micro-colony, thereby showing that they were neither mini cells, spores nor dormant forms of mycobacteria. The freshly diagnosed pulmonary tuberculosis patients' sputum samples, which are known for the prevalence of oxidative stress conditions, also contained short cells at the same proportion as that in the mid-log phase population. The probable physiological significance of the generation of the short cells through unusually deviated asymmetric cell division is discussed

A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity

Diadenosine polyphosphates, Ap_(2–7)A, which contain two adenosines in a 5′,5′ linkage through phosphodiester bonds involving 2–7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of <b><u>a</u></b>symmetric <b><u>c</u></b>onstriction during <b><u>d</u></b>ivision (ACD) in 20–30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70–90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap₆A. Synthetic Ap₆A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap₆A in the CCS. Thus, the present work reveals a novel role for Ap₆A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population

A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity

Diadenosine polyphosphates, Ap_(2–7)A, which contain two adenosines in a 5′,5′ linkage through phosphodiester bonds involving 2–7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of <b><u>a</u></b>symmetric <b><u>c</u></b>onstriction during <b><u>d</u></b>ivision (ACD) in 20–30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70–90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap₆A. Synthetic Ap₆A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap₆A in the CCS. Thus, the present work reveals a novel role for Ap₆A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population

A Symmetric Molecule Produced by Mycobacteria Generates Cell-Length Asymmetry during Cell-Division and Thereby Cell-Length Heterogeneity

Diadenosine polyphosphates, Ap_(2–7)A, which contain two adenosines in a 5′,5′ linkage through phosphodiester bonds involving 2–7 phosphates, regulate diverse cellular functions in all organisms, from bacteria to humans, under normal and stress conditions. We had earlier reported consistent occurrence of <b><u>a</u></b>symmetric <b><u>c</u></b>onstriction during <b><u>d</u></b>ivision (ACD) in 20–30% of dividing mycobacterial cells in culture, irrespective of different growth media, implying exogenous action of some factor of mycobacterial origin. Consistent with this premise, concentrated culture supernatant (CCS), but not the equivalent volume-wise concentrated unused medium, dramatically enhanced the ACD proportion to 70–90%. Mass spectrometry and biochemical analyses of the bioactive fraction from CCS revealed the ACD-effecting factor to be Ap₆A. Synthetic Ap₆A showed a mass spectrometry profile, biochemical characteristics, and bioactivity identical to native Ap₆A in the CCS. Thus, the present work reveals a novel role for Ap₆A in generating cell-length asymmetry during mycobacterial cell-division and thereby cell-length heterogeneity in the population

Complete identity and expression of StfZ, the cis-antisense RNA to the mRNA of the cell division gene ftsZ, in Escherichia coli

Bacteria regulate FtsZ protein levels through transcriptional and translational mechanisms for proper cell division. A cis-antisense RNA, StfZ, produced from the ftsA-ftsZ intergenic region, was proposed to regulate FtsZ level in Escherichia coli. However, its structural identity remained unknown. In this study, we determined the complete sequence of StfZ and identified the isoforms and its promoters. We find that under native physiological conditions, StfZ is expressed at a 1:6 ratio of StfZ:ftsZ mRNA at all growth phases from three promoters as three isoforms of 366, 474, and 552 nt RNAs. Overexpression of StfZ reduces FtsZ protein level, increases cell length, and blocks cell division without affecting the ftsZ mRNA stability. We did not find differential expression of StfZ under the stress conditions of heat shock, cold shock, or oxidative stress, or at any growth phase. These data indicated that the cis-encoded StfZ antisense RNA to ftsZ mRNA may be involved in the fine tuning of ftsZ mRNA levels available for translation as per the growth-phase-specific requirement at all phases of growth and cell division

Loss of GdpP Function in Staphylococcus aureus Leads to β-Lactam Tolerance and Enhanced Evolution of β-Lactam Resistance

Infections caused by Staphylococcus aureus are a leading cause of mortality. Treating infections caused by S. aureus is difficult due to resistance against most traditional antibiotics, including β-lactams. We previously reported the presence of mutations in gdpP among S. aureus strains that were obtained by serial passaging in β-lactam drugs. Similar mutations have recently been reported in natural S. aureus isolates that are either nonsusceptible or resistant to β-lactam antibiotics. gdpP codes for a phosphodiesterase that cleaves cyclic-di-AMP (CDA), a newly discovered second messenger. In this study, we sought to identify the role of gdpP in β-lactam resistance in S. aureus. Our results showed that gdpP-associated mutations caused loss of phosphodiesterase function, leading to increased CDA accumulation in the bacterial cytosol. Deletion of gdpP led to an enhanced ability of the bacteria to withstand a β-lactam challenge (2 to 3 log increase in bacterial CFU) by promoting tolerance without enhancing MICs of β-lactam antibiotics. Our results demonstrated that increased drug tolerance due to loss of GdpP function can provide a selective advantage in acquisition of high-level β-lactam resistance. Loss of GdpP function thus increases tolerance to β-lactams that can lead to its therapy failure and can permit β-lactam resistance to occur more readily