Although reports of flow cytometry (FCM) applied to bacterial analysis are increasing, studies of FCM related to human cells still vastly outnumber other reports. However, current advances in FCM combined with a new generation of cellular reporter probes have made this technique suitable for analyzing physiological responses in bacteria. We review how FCM has been applied to characterize distinct physiological conditions in bacteria including responses to antibiotics and other cytotoxic chemicals and physical factors, pathogen-host interactions, cell differentiation during biofilm formation, and the mechanisms governing development pathways such as sporulation. Since FCM is suitable for performing studies at the single-cell level, we describe how this powerful technique has yielded invaluable information about the heterogeneous distribution of differently and even specialized responding cells and how it may help to provide insights about how cell interaction takes place in complex structures, such as those that prevail in bacterial biofilms.

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.Since the mid-1950s, microbiologists have been aware of mutations occurring in nondividing populations of cells (22,29). The formation of these mutants was alternatively termed "starvation-associated mutagenesis" (29), "adaptive mutation" (8), or "stationary-phase mutagenesis" (13). Recently, variations of this phenomenon have been investigated with Escherichia coli (8,29,38), Pseudomonas (33), Bacillus subtilis (41), and the eukaryotic yeast Saccharomyces cerevisiae (15,40). These phenomena reveal that starving populations of cells can acquire mutations favoring growth after the application of selection.While the phenomenon of stationary-phase mutation is widespread, it is clear that the mechanism(s) by which it arises varies from organism to organism. To date, the most favored system for studying adaptive or stationary-phase mutagenesis is the RecA-dependent E. coli FC40 system investigated by, among others, the laboratories of Cairns, Foster, Rosenberg, and Roth (7,8,11,18). Recent results strongly suggest that this mutagenesis is the result of gene amplification followed by mutation in a transiently growing population of cells (18). In light of this, we chose to investigate the possibility that transiently growing cells may play a role in a B. subtilis system containing three chromosomal point mutations. The phenomenon of transcriptional mutagenesis, or retromutagenesis, whereby RNA polymerase bypasses an unrepaired DNA lesion or otherwise produces an altered mRNA, which is then translated into a protein of altered function, could provide a transient growth advantage for the cell. This mechanism has been proposed for other model systems, including eukaryotes (6, 9).We have previously shown the existence of one such mutagenic phenomenon occurring during stationary phase in B. subtilis cells starved for amino acids (41). This mutagenic process appears to enhance the survivability of cell populations undergoing nutritional stress. In brief, isogenic strains of B. subtilis carrying three amino acid auxotrophies conferred by hisC952 (amber), metB5 (ochre), and leuC427 (missense) are incubated on medium lacking one of the required amino acids. After several days of incubati...

Abstract. 1. Immune priming refers to improved protection of the host after a second encounter with the same parasite or pathogen. This phenomenon is similar to that of adaptive immunity in vertebrates.2. There is evidence to suggest that this improved protection can be species/ strain-specific and can protect organisms for a lifetime. These two attributes, along with a biphasic immune response, are essential characteristics of immune priming and form the basis for the effectiveness of resistance to parasites and pathogens.3. This paper considers the effect of immune priming within and across generations, the influence of a heterologous challenge during immune priming and the importance of testing the immune response with natural pathogens.4. The analysis presented takes into account the multifaceted nature of the invertebrate immune response. The lack of evidence suggesting that the bacterial microbiome plays a complementary role in the immune priming outcome is discussed.5. Finally, the cost of immune priming is explored. This is a poorly investigated issue, which could help to explain why there is a paucity of evidence in support of immune priming.

A reversion assay system previously implemented to demonstrate the existence of adaptive or stationaryphase-induced mutagenesis in Bacillus subtilis was utilized in this report to study the influence of the mismatch DNA repair (MMR) system on this type of mutagenesis. Results revealed that a strain deficient in MutSL showed a significant propensity to generate increased numbers of stationary-phase-induced revertants. These results suggest that absence or depression of MMR is an important factor in the mutagenesis of nongrowing B. subtilis cells because of the role of MMR in repairing DNA damage. In agreement with this suggestion, a significant decrease in the number of adaptive revertant colonies, for the three markers tested, occurred in B. subtilis cells which overexpressed a component of the MMR system. Interestingly, the single overexpression of mutS, but not of mutL, was sufficient to decrease the level of adaptive mutants in the reversion assay system of B. subtilis. The results presented in this work, as well as in our previous studies, appear to suggest that an MMR deficiency, putatively attributable to inactivation or saturation with DNA damage of MutS, may occur in a subset of B. subtilis cells that differentiate into the hypermutable state.Adaptive or stationary-phase-induced mutagenesis occurs in nondividing cells during prolonged nonlethal selective pressure, e.g., starvation for an essential amino acid (25). While most of the research has involved Escherichia coli model systems, similar observations have been made in other prokaryotes (14, 30) as well as in eukaryotic organisms (8). The most widely studied system thus far has been the FЈ lac frameshift-reversion construct of E. coli (25). In this system it has been demonstrated that generation of Lac ϩ stationary-phaseassociated revertants is dependent on (i) a functional Rec system (10), (ii) FЈ transfer functions (6), and (iii) a component(s) of the SOS system (18, 19). In addition, both DNA polymerase III and the SOS-regulated DNA polymerase IV (19) have been shown to be responsible for the synthesis of errors that lead to these mutations (for review of the SOS regulon see reference 33). More recent evidence demonstrates that the mutations generated by this lac system during stationary phase are the result of actual cell growth and amplification of the plasmid-borne gene that is followed by SOS-induced mutagenesis and selection (11,28).The existence of stationary-phase-induced mutagenesis was recently demonstrated in Bacillus subtilis following the utilization of a reversion assay system (30). In contrast to the FЈ lac system of E. coli, this type of mutagenesis in B. subtilis is not dependent upon a functional RecA protein (i.e., recombination or the activation of type 1 SOS functions [35] was not required). Moreover, it was also demonstrated that generation of B. subtilis adaptive mutants did not require a functional B factor (RNA polymerase B controls the general stress response in B. subtilis [34]). However, one of the most relevant outcomes...

The major photoproduct in UV-irradiated spore DNA is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). An important determinant of the high UV resistance of Bacillus subtilis spores is the accurate in situ reversal of SP during spore germination by the DNA repair enzyme SP lyase. To study the molecular aspects of SP lyase-mediated SP repair, the cloned B. subtilis splB gene was engineered to encode SP lyase with a molecular tag of six histidine residues at its amino terminus. The engineered six-His-tagged SP lyase expressed from the amyE locus restored UV resistance to spores of a UV-sensitive mutant B. subtilis strain carrying a deletion-insertion mutation which removed the entire splAB operon at its natural locus and was shown to repair SP in vivo during spore germination. The engineered SP lyase was purified both from dormant B. subtilis spores and from an Escherichia colioverexpression system by nickel-nitrilotriacetic acid (NTA) agarose affinity chromatography and was shown by Western blotting, UV-visible spectroscopy, and iron and acid-labile sulfide analysis to be a 41-kDa iron-sulfur (Fe-S) protein, consistent with its amino acid sequence homology to the 4Fe-4S clusters in anaerobic ribonucleotide reductases and pyruvate-formate lyases. SP lyase was capable of reversing SP from purified SP-containing DNA in an in vitro reaction either when present in a cell-free extract prepared from dormant spores or after purification on nickel-NTA agarose. SP lyase activity was dependent upon reducing conditions and addition ofS-adenosylmethionine as a cofactor.

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.The generation of mutations has been traditionally ascribed to spontaneous processes affecting actively growing, dividing cells. Nevertheless, by the mid-1950s, several reports describing mutagenesis in nondividing cells of bacteria, plants, flies, and fungi appeared in the scientific literature (reference 36 and references therein). Much of the initial characterization of this process in bacteria took place in the laboratory of Francis Ryan, who observed Escherichia coli mutants capable of synthesizing histidine arising from his mutant (auxotrophic) cells undergoing prolonged starvation (36) while cell turnover remained undetectable, and DNA replication slowed with increasing time (26). Renewed interest in adaptive mutation was generated when Cairns and coworkers published their work on the generation of Lac ϩ reversions in E. coli cells unable to use the lactose provided as the sole carbon source in a minimal medium (6). This work demonstrated that adaptive mutations can arise as a result of stress rather than from selection of preexisting mutations. The generation of stress-induced Lac ϩ reversions, assayed via a plasmid-borne system, has been studied intensively by several laboratories (reviewed in references 13, 15, and 34; 32) and is dependent on activation of the SOS and/or stress responses. Further studies have also suggested that a subpopulation within the Lac Ϫ stressed cells engage in an exquisitely regulated transient state of hypermutation limited in time and to DNA sites near double-stranded DNA breaks (reviewed in reference 15). Collectively, the results from studies on this system have provided interesting insights into the acquisition of beneficial mutations and demonstrated the role of several genetic factors in the adaptive mutation phenomenon.

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 stationaryphase-induced mutagenesis in this soil microorganism.Adaptive or stationary-phase mutagenesis can be defined as those mutations that permit organisms to grow and divide in response to natural or artificial selection (5) and that occur in nondividing cells during prolonged nonlethal selective pressure, e.g., starvation for an essential amino acid (32). Although this type of mutagenesis was first described in Escherichia coli (7), additional examples of adaptive mutagenesis in other prokaryotes (21, 41) and in eukaryotic organisms (14) have been published. In some cases, these mutations occurred in the absence of specific selection but in response to starvation (11). Regardless of the organisms utilized and the name used, these types of mutations and the processes that generate them are of real interest with respect to evolution and the generation of diversity across all domains of life.Studies with the FЈ lac frameshift reversion construct of E. coli (32) have demonstrated that the generation of Lac ϩ stationary-phase-associated revertants required functional recombination (15), as well as component(s) of the SOS system (24, 25). Further evidence suggests that the mutations generated by this lac system during stationary phase may also be the result of amplification of the plasmid-borne gene followed by SOS-induced mutagenesis and selection (18,38). Recent studies have demonstrated that DNA double-strand-break repair, in addition to the SOS DNA damage response and the error-prone DNA polymerase, are necessary for stress-induced reversion of the E. col...

SummaryIn 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 nongrowing sporulating cells, Mfd played an antimutagenic role as its absence promoted UV-induced mutagenesis through a pathway involving YqjH/YqjWmediated 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.