Eduardo A. Robleto

Novel Role of mfd: Effects on Stationary-Phase Mutagenesis in Bacillus subtilis

Previously, using a chromosomal reversion assay system, we established that an adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under stress, and we demonstrated that multiple mechanisms are involved in generating these mutations (41, 43). In an attempt to delineate how these mutations are generated, we began an investigation into whether or not transcription and transcription-associated proteins influence adaptive mutagenesis. In B. subtilis, the Mfd protein (transcription repair coupling factor) facilitates removal of RNA polymerase stalled at transcriptional blockages and recruitment of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An association between Mfd mutagenesis and aspects of transcription is discussed.

Genetic analysis of the AdnA regulon in Pseudomonas fluorescens: nonessential role of flagella in adhesion to sand and biofilm formation.

AdnA is a transcription factor in Pseudomonas fluorescens that affects flagellar synthesis, biofilm formation, and sand adhesion. To identify the AdnA regulon, we used a promoterless Tn5-lacZ element to study the phenotypes of insertion mutants in the presence and absence of AdnA. Of 12,000 insertions, we identified seven different putative open reading frames (ORFs) activated by AdnA (named aba for activated by AdnA). aba120 and aba177 showed homology to flgC and flgI, components of the basal body of the flagella in Pseudomonas aeruginosa. Two other insertions, aba18 and aba51, disrupted genes affecting chemotaxis. The mutant loci aba160 (possibly affecting lipopolysaccharide synthesis) and aba175 (unknown function) led to loss of flagella. The mutant bearing aba203 became motile when complemented with adnA, but the mutated gene showed no similarity to known genes. Curiously, aba18, aba51, aba160, and aba203 mutants formed biofilms even in the absence of AdnA, suppressing the phenotype of the adnA deletion mutant. The combined findings suggest that flagella are nonessential for sand attachment or biofilm formation. Sequence and promoter analyses indicate that AdnA affects at least 23 ORFs either directly or by polar effects. These results support the concept that AdnA regulates cell processes other than those directly related to flagellar synthesis and define a broader cadre of genes in P. fluorescens than that described so far for its homolog, FleQ, in P. aeruginosa.

Transcription-Associated Mutation in Bacillus subtilis Cells under Stress

Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427, under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu(+) reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.

Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis

Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism.

The adnA transcriptional factor affects persistence and spread of Pseudomonas fluorescens under natural field conditions.

ABSTRACT A soil plot was inoculated with a mixture of Pseudomonas fluorescens Pf0-2, the wild type, and Pf0-5a, a Tn5insertion mutant in adnA, at 7.84 log CFU/g of soil. Over a period of 231 days, culturable populations of both strains were measured at selected times below and away from the point of inoculation. Pf0-5a did not spread as fast and attained significantly lower populations than Pf0-2. At sample depths below the inoculation site, the adnA mutant showed a significant decrease in CFU/g of soil as compared to Pf0-2. Pf0-2 was first detected at the 1.5-cm annular site at 3 days after inoculation, whereas Pf0-5a required 7 days to travel the same distance. At this distance, the wild-type strain could be detected at a 21.5- to 25-cm depth, whereas Pf0-5a could be detected only as deep as 15.5 to 18 cm. At 4.5 cm from the site of inoculation and in soil fractions corresponding to 13 to 18 cm, Pf0-2 was the only strain detected. These results suggest that the transcription factor AdnA provides a fitness advantage in P. fluorescens, allowing it to spread and survive in soil under field conditions.

Transcriptional coupling of DNA repair in sporulating Bacillus subtilis cells.

In conditions of halted or limited genome replication, like those experienced in sporulating cells of Bacillus subtilis, a more immediate detriment caused by DNA damage is altering the transcriptional programme that drives this developmental process. Here, we report that mfd, which encodes a conserved bacterial protein that mediates transcription‐coupled DNA repair (TCR), is expressed together with uvrA in both compartments of B. subtilis sporangia. The function of Mfd was found to be important for processing the genetic damage during B. subtilis sporulation. Disruption of mfd sensitized developing spores to mitomycin‐C (M‐C) treatment and UV‐C irradiation. Interestingly, in non‐growing sporulating cells, Mfd played an anti‐mutagenic role as its absence promoted UV‐induced mutagenesis through a pathway involving YqjH/YqjW‐mediated translesion synthesis (TLS). Two observations supported the participation of Mfd‐dependent TCR in spore morphogenesis: (i) disruption of mfd notoriously affected the efficiency of B. subtilis sporulation and (ii) in comparison with the wild‐type strain, a significant proportion of Mfd‐deficient sporangia that survived UV‐C treatment developed an asporogenous phenotype. We propose that the Mfd‐dependent repair pathway operates during B. subtilis sporulation and that its function is required to eliminate genetic damage from transcriptionally active genes.

Error-Prone Processing of Apurinic/Apyrimidinic (AP) Sites by PolX Underlies a Novel Mechanism That Promotes Adaptive Mutagenesis in Bacillus subtilis

In growing cells, apurinic/apyrimidinic (AP) sites generated spontaneously or resulting from the enzymatic elimination of oxidized bases must be processed by AP endonucleases before they compromise cell integrity. Here, we investigated how AP sites and the processing of these noncoding lesions by the AP endonucleases Nfo, ExoA, and Nth contribute to the production of mutations (hisC952, metB5, and leuC427) in starved cells of the Bacillus subtilis YB955 strain. Interestingly, cells from this strain that were deficient for Nfo, ExoA, and Nth accumulated a greater amount of AP sites in the stationary phase than during exponential growth. Moreover, under growth-limiting conditions, the triple nfo exoA nth knockout strain significantly increased the amounts of adaptive his, met, and leu revertants produced by the B. subtilis YB955 parental strain. Of note, the number of stationary-phase-associated reversions in the his, met, and leu alleles produced by the nfo exoA nth strain was significantly decreased following disruption of polX. In contrast, during growth, the reversion rates in the three alleles tested were significantly increased in cells of the nfo exoA nth knockout strain deficient for polymerase X (PolX). Therefore, we postulate that adaptive mutations in B. subtilis can be generated through a novel mechanism mediated by error-prone processing of AP sites accumulated in the stationary phase by the PolX DNA polymerase.

Colonization strategies of Pseudomonas fluorescens Pf0-1: activation of soil-specific genes important for diverse and specific environments

BackgroundPseudomonas fluorescens is a common inhabitant of soil and the rhizosphere environment. In addition to potential applications in biocontrol and bioremediation, P. fluorescens is of interest as a model for studying bacterial survival and fitness in soil. A previous study using in vivo expression technology (IVET) identified 22 genes in P. fluorescens Pf0-1 which are up-regulated during growth in Massachusetts loam soil, a subset of which are important for fitness in soil. Despite this and other information on adaptation to soil, downstream applications such as biocontrol or bioremediation in diverse soils remain underdeveloped. We undertook an IVET screen to identify Pf0-1 genes induced during growth in arid Nevada desert soil, to expand our understanding of growth in soil environments, and examine whether Pf0-1 uses general or soil type-specific mechanisms for success in soil environments.ResultsTwenty six genes were identified. Consistent with previous studies, these genes cluster in metabolism, information storage/processing, regulation, and ‘hypothetical’, but there was no overlap with Pf0-1 genes induced during growth in loam soil. Mutation of both a putative glutamine synthetase gene (Pfl01_2143) and a gene predicted to specify a component of a type VI secretion system (Pfl01_5595) resulted in a decline in arid soil persistence. When examined in sterile loam soil, mutation of Pfl01_5595 had no discernible impact. In contrast, the Pfl01_2143 mutant was not impaired in persistence in sterile soil, but showed a significant reduction in competitive fitness.ConclusionsThese data support the conclusion that numerous genes are specifically important for survival and fitness in natural environments, and will only be identified using in vivo approaches. Furthermore, we suggest that a subset of soil-induced genes is generally important in different soils, while others may contribute to success in specific types of soil. The importance of glutamine synthetase highlights a critical role for nitrogen metabolism in soil fitness. The implication of Type 6 secretion underscores the importance of microbial interactions in natural environments. Understanding the general and soil-specific genes will greatly improve the persistence of designed biocontrol and bioremediation strains within the target environment.

Roles of Endonuclease V, Uracil-DNA Glycosylase, and Mismatch Repair in Bacillus subtilis DNA Base-Deamination-Induced Mutagenesis

The disruption of ung, the unique uracil-DNA-glycosylase-encoding gene in Bacillus subtilis, slightly increased the spontaneous mutation frequency to rifampin resistance (Rif(r)), suggesting that additional repair pathways counteract the mutagenic effects of uracil in this microorganism. An alternative excision repair pathway is involved in this process, as the loss of YwqL, a putative endonuclease V homolog, significantly increased the mutation frequency of the ung null mutant, suggesting that Ung and YwqL both reduce the mutagenic effects of base deamination. Consistent with this notion, sodium bisulfite (SB) increased the Rif(r) mutation frequency of the single ung and double ung ywqL strains, and the absence of Ung and/or YwqL decreased the ability of B. subtilis to eliminate uracil from DNA. Interestingly, the Rif(r) mutation frequency of single ung and mutSL (mismatch repair [MMR] system) mutants was dramatically increased in a ung knockout strain that was also deficient in MutSL, suggesting that the MMR pathway also counteracts the mutagenic effects of uracil. Since the mutation frequency of the ung mutSL strain was significantly increased by SB, in addition to Ung, the mutagenic effects promoted by base deamination in growing B. subtilis cells are prevented not only by YwqL but also by MMR. Importantly, in nondividing cells of B. subtilis, the accumulations of mutations in three chromosomal alleles were significantly diminished following the disruption of ung and ywqL. Thus, under conditions of nutritional stress, the processing of deaminated bases in B. subtilis may normally occur in an error-prone manner to promote adaptive mutagenesis.

Transcriptional De-Repression and Mfd Are Mutagenic in Stressed Bacillus subtilis Cells

In recent years, it has been proposed that conflicts between transcription and active chromosomal replication engender genome instability events. Furthermore, transcription elongation factors have been reported to prevent conflicts between transcription and replication and avoid genome instability. Here, we examined transcriptional de-repression as a genetic diversity-producing agent and showed, through the use of physiological and genetic means, that transcriptional de-represssion of a leuC defective allele leads to accumulation of Leu+ mutations. We also showed, by using riboswitches that activate transcription in conditions of tyrosine or methionine starvation, that the effect of transcriptional de-repression of the leuC construct on the accumulation of Leu+ mutations was independent of selection. We examined the role of Mfd, a transcription elongation factor involved in DNA repair, in this process and showed that proficiency of this factor promotes mutagenic events. These results are in stark contrast to previous reports in Escherichia coli, which showed that Mfd prevents replication fork collapses. Because our assays place cells under non-growing conditions, by starving them for two amino acids, we surmised that the Mfd mutagenic process associated with transcriptional de-repression does not result from conflicts with chromosomal replication. These results raise the interesting concept that transcription elongation factors may serve two functions in cells. In growing conditions, these factors prevent the generation of mutations, while in stress or non-growing conditions they mediate the production of genetic diversity.