When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase ?-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.
Transcription and translation of mRNA's are coordinated processes in bacteria. We have previously shown that a mutant form of EF-Tu (Gln125Arg) in Salmonella Typhimurium with a reduced affinity for aa-tRNA, causes ribosome pausing, resulting in an increased rate of RNase E-mediated mRNA cleavage, causing extremely slow growth, even on rich medium. The slow growth phenotype is reversed by mutations that reduce RNase E activity. Here we asked whether the slow growth phenotype could be reversed by overexpression of a wild-type gene. We identified spoT (encoding ppGpp synthetase/hydrolase) as a gene that partially reversed the slow growth rate when overexpressed. We found that the slow-growing mutant had an abnormally high basal level of ppGpp that was reduced when spoT was overexpressed. Inactivating relA (encoding the ribosome-associated ppGpp synthetase) also reduced ppGpp levels and significantly increased growth rate. Because RelA responds specifically to deacylated tRNA in the ribosomal A-site this suggested that the tuf mutant had an increased level of deacylated tRNA relative to the wild-type. To test this hypothesis we measured the relative acylation levels of 4 families of tRNAs and found that proline isoacceptors were acylated at a lower level in the mutant strain relative to the wild-type. In addition, the level of the proS tRNA synthetase mRNA was significantly lower in the mutant strain. We suggest that an increased level of deacylated tRNA in the mutant strain stimulates RelA-mediated ppGpp production, causing changes in transcription pattern that are inappropriate for rich media conditions, and contributing to slow growth rate. Reducing ppGpp levels, by altering the activity of either SpoT or RelA, removes one cause of the slow growth and reveals the interconnectedness of intracellular regulatory mechanisms.
Intermediate filament (IF)-like cytoskeleton emerges as a versatile tool for cellular organization in all kingdoms of life, underscoring the importance of mechanistically understanding its diverse manifestations. We showed previously that, in Streptomyces (a bacterium with a mycelial lifestyle similar to that of filamentous fungi, including extreme cell and growth polarity), the IF protein FilP confers rigidity to the hyphae by an unknown mechanism. Here, we provide a possible explanation for the IF-like function of FilP by demonstrating its ability to self-assemble into a cis-interconnected regular network in vitro and its localization into structures consistent with a cytoskeletal network in vivo. Furthermore, we reveal that a spatially restricted interaction between FilP and DivIVA, the main component of the Streptomyces polarisome complex, leads to formation of apical gradients of FilP in hyphae undergoing active tip extension. We propose that the coupling between the mechanism driving polar growth and the assembly of an IF cytoskeleton provides each new hypha with an additional stress-bearing structure at its tip, where the nascent cell wall is inevitably more flexible and compliant while it is being assembled and matured. Our data suggest that recruitment of cytoskeleton around a cell polarity landmark is a broadly conserved strategy in tip-growing cells.
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