Genetic variability among archival cultures ofSalmonella typhimurium

Kelly Edwards a, Irina Linetsky a, Christopher Hueser a, Abraham Eisenstark a;b;*a Cancer Research Center, 3501 Berrywood Drive, Columbia, MO 65201, USA

b Division of Biological Sciences, and Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211, USA

Received 21 November 2000; received in revised form 2 March 2001; accepted 12 April 2001

First published online 4 May 2001

Abstract

The existence in our laboratory of over 10 000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33^46 years offers anopportunity for evolutionary and mutational studies. In each of 77 vials examined, 103^105 colony forming units per vial were recovered(less than 0.01% of the original population) even after decades of undisturbed storage. Considerable genetic variability was observed in thesepopulations. Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutationalreversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, were measured. ß 2001 Federation of European Microbiological Societies. Published by ElsevierScience B.V. All rights reserved.

Keywords: Genetic variability ; Salmonella typhimurium

1. Introduction

Starting in the late 1940s, Demerec and associates atCold Spring Harbor Laboratory and Brookhaven Nation-al Laboratory stocked an extensive collection of auxotro-phic mutants primarily for the purpose of mapping of theSalmonella typhimurium LT2 chromosome [1^3]. Over10 000 of these original sealed agar stab cultures (0.5 mlper agar stab vial) are now curated in our laboratory.Upon initial inoculation, low reversion rates of less than1 per 108 cells were recorded, indicating genetic stability ofeach auxotroph placed in the collection. While ¢ve dec-ades is a short span in evolutionary history, initial resultsshow considerable genetic and phenotypic variationamong the survivors. In this report, di¡erences in chromo-some fragment sizes as determined by pulsed-¢eld gel elec-trophoresis (PFGE), extensive mutational reversions fromauxotrophy to prototrophy, and di¡erences in protein pro-¢les as seen by Coomassie-stained sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS^PAGE) are de-scribed.

2. Materials and methods

2.1. Bacterial strains and microbiological methods

Standard microbiological [4,5] procedures were used.The complete listing of strains and relevant genotypes ofeach of the 77 strains used in this study can be found athttp://www.cancerresearchcenter.org/table1.html.

2.2. Treatment of samples before re-growth

When the entire content of a vial is used for re-growth,that specimen loses its antiquity. Thus, for many of thevials, only a portion of the content was removed, and theremainder was resealed for further observation. Uponopening a vial, a segment of the stab was removed andthe content placed into a tube containing a small amountof sterile phosphate bu¡ered saline (PBS). In all of thevials that showed no sign of drying out, cells were recov-ered, usually 103^105 colony forming units (cfu) per vial.

Bacterial strains were tested for identity as S. typhimu-rium with P22 phage and with group B Salmonella O anti-gen speci¢c antiserum. Pre-growth samples were sus-pended in PBS and then cells were plated for cfu onappropriate media to determine any heterogeneity of col-

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 1 8 5 - 9

* Corresponding author.

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www.fems-microbiology.org

ony morphology and the ratio of auxotrophs to proto-trophs. When inoculated decades ago, reversion to proto-trophy was not detectable or less than 1 per 108 cells.

In addition to characterizing S. typhimurium cells, sam-ples were tested for the presence of viable phage by platingthe supernatant from a culture on a range of Salmonellahosts. Phages were present in a few samples of superna-tants, but have yet to be compared with known phages.

2.3. PFGE genome sizing

Isolates were cultured to exponential phase in trypticsoy or Luria^Bertani (LB) broth. Genomic DNA was iso-lated in agarose blocks according to standard methodsand PFGE was performed on selected strains [6]. Agaroseslices containing enzymatically digested DNA were placedinto the wells of the SeaKem gel and sealed in place withwarm 1% agarose in TBE bu¡er. The Lambda Laddermolecular mass marker (Bio-Rad, Hercules, CA, USA)was used to measure accurate sizing. Pulsed-¢eld electro-phoresis was performed at 200 V for 24 h with pulse timeramped from 5 to 50 s and bu¡er temperature maintainedat 14³C using the CHEF Mapper (Bio-Rad, Hercules, CA,USA). The gels were stained with ethidium bromide andscanned under UV light.

2.4. Test of reversion from auxotrophy to prototrophy

Reversion to wild-type was scored in these cultures,each of which initially had a requirement for a speci¢camino acid, nucleotide or vitamin. Samples were takendirectly from vials, serially diluted, and plated on appro-priate media to obtain a ratio of auxotrophs to proto-trophs, as well as to determine the number of survivors.Among 102^105 surviving cfu per vial, prototrophs wereobserved in 49 of the 77 samples. These ratios were alsomeasured after transfer to LB and growth for 18 h at37³C. The ratios of auxotrophs to prototrophs basicallyremained the same.

2.5. Protein variability

Altered protein patterns by SDS^PAGE were measuredusing standard procedure [7]. Coomassie stain was appliedand destained after electrophoresis.

3. Results

Before opening a vial, the color of the agar was noted asa clue to metabolic changes. The most obvious changeshave been observed among the auxotrophs requiring aro-matic amino acids.

3.1. PFGE

PFGE assays, using AvrII restriction enzyme, revealedtwo striking features. First, there was heterogeneity insizes of fragments generated by restriction enzyme diges-tion (Fig. 1). Another di¡erence between the aged isolatesand the reference non-archival control (as well as that ofgenomes recorded in publications) [8^11] is the absence ofa 180-kb band when cut with restriction enzyme AvrII.Only one of our archival samples had this band of ca.180 kb (strain 1600), resulting in di¡erences among sizesfor the total genome.

There are two explanations for the absence of this band.Either the genes within the 180-kb band may be missing orthe particular restriction fragment, but not the cognatesites, may have moved to another position in the genome.In the ongoing S. typhimurium sequencing project [12], the180-kb fragment after AvrII treatment is designatedSTMD2. This segment contains many inverted repeatsand ¢nal interpretation and annotation is still under wayat the sequencing facility at Washington University. Ob-servations of phenotypes would indicate that at least someif not all of these genes known to be on the STMD2 frag-ment were present. The cells were still motile (i.e., £agellawere still present indicating the presence of £iA and £iBgenes). The strains do not require additional amino acids,and remain recombination pro¢cient (data not shown).Thus, our current view is that one of the AvrII sites isno longer present, and that the genes in the STMD2

Fig. 1. Loss of large restriction fragment(s) following archival storage asshown by PFGE. Estimated changes in genome sizes in kb are indicatedin parentheses following isolate numbers. Lane 1: 1594 his-2555 (4655);lane 2: 1596 his-2555 (4196); lane 3: 1600 his-143 (4800); lane 4: 1602(4633); lane 5: 1615 his-2543 (4655); lane 6: 1619 his-2553 (4655); lane7: 1639 his-142 (4655); lane 8: 1656 his-68 (4775); lane 9: 1657 his-2122(4655); lane 10: kb 1670 his-2550 (4672); lane 11: 1671 his-2550 (4672);lane 12: 1674 his-2550 (5846); lane 13: 1700 his-314 (4655). Molecularmass marker is Lambda Ladder.

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band are now in another region of the genome. Fig. 1demonstrates the genomic heterogeneity of several archivalS. typhimurium.

A striking di¡erence observed is that several of the ar-chival cultures had a common origin, i.e., decades ago,several vials were inoculated from the same mutant colonyand then stored. Yet, there was a di¡erence of over 450 kbin genome size as shown in his-2555 (strains 1594 and1596) and his-2550 (strains 1670, 1671, 1674). Strain1674 had an increased genome length of over 1000 kbover 1670 and 1671.

Other investigators [8^11] have also reported variabilityin PFGE restriction enzyme patterns and genome sizes indi¡erent strains of Salmonella. It will be of interest todetermine which genes are missing in the shortest genome(strain 1596) as well as what extra genes are present in thelongest genome (strain 1674). As a control to checkwhether there might be diversity among non-archival sib-lings, PFGE was performed on cells from 12 di¡erentcolonies of strain 1602 after digestion with AvrII enzyme.No di¡erences were detected in genome size among these12 sibling colonies (data not shown).

3.2. Reversion from auxotrophy to prototrophy

Of the 77 originally auxotrophic archival strains, only28 revealed no evidence of reversion to prototrophy (i.e.,no colonies on minimal agar plates). In 32 cultures, cfuwere approximately the same on LB as on minimal agarplates, indicating that the surviving populations consistedof prototrophs only. The remaining cultures had substan-tially lower counts on minimal plates than on LB plates,thus indicating a mixture of prototrophs and auxotrophs.

Of particular interest, several of the archival culturesstemmed from the same isolate stored in multiple vials.There was diversity in the quantitative extent of reversionfrom auxotrophy to prototrophy. Thus, the populationchanges took di¡erent courses even when the samplesstemmed from the same parent cultures. A census of pro-totrophs and auxotrophs recovered in the 77 archival cul-tures may be found in detail at www.cancerresearchcen-ter.org.

3.3. Heterogeneity in protein content

Having observed variability in DNA by PFGE, varia-bility in protein expression was anticipated as well. Inparticular, we queried whether reduction in genome sizewould correlate with protein content. SDS^PAGE andCoomassie staining, indeed, revealed extensive heterogene-ity among these archival cultures. Examples of patterndi¡erences are noted in Fig. 2. However, the extent ofdiversity was di¤cult to assess without knowledge of spe-ci¢c proteins present or absent in various bands. In gen-eral, only 23 of the strains showed di¡erences from thereference strain. These strains (1596, 1601, 1614, 1671,

1672, 1700, 1737 and 1745) might be worth further proteinanalysis because of extensive di¡erences. In particular,proteins missing in strain 1596 need to be determined tosee if this correlates with the loss of genome (Fig. 2).

3.4. No common genetic change detected

It is important to note that we detected no commonpattern of deviation from the non-archival reference strainin all of the 77 samples examined by the three methods inthis investigation. The best evidence of a uniform geneticchange upon long storage is the loss of the 180-kb band inall but one of the archival strains examined. Also, 49 ofthe 77 strains mutated from auxotrophy to prototrophy,and this reversion of phenotype may be a factor in surviv-al. Of these 49, a few also showed changes in PFGE andprotein assays.

4. Discussion

Laboratory records for the years 1954^1966 show thatfor each of the 77 strains tested reversions were eitherundetected or less than 1038 when tested before inocula-tion to vials. Since the purpose of collecting auxotrophswas for intra- and intergenic mapping, low reversion rateswere necessary for scoring recombinants in genetic crosses.

4.1. Mutation and ¢tness studies by other investigators

Viable bacteria were recovered after decades of con¢ne-ment although survivors di¡ered considerably from theiroriginal strains. Questions may be addressed: (a) underthese conditions of restricted nutrition and cell divisiondid these descendants shed (or re-arrange) unnecessaryexcess genetic baggage; (b) was there a `use it or lose it'motif whereby there would be a selective preference of

Fig. 2. Diversity of protein patterns among archival strains. Lanes 1^6show strains 1699, 1700, 1701, 1702, 1704, 1706. Lane 7: Bio-Rad sizestandards.

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cells with `survival' genes as described by Koch; [13]; or,(c) were the changes scattered, thus resulting in randomdiversity in the population?

While ¢ve decades in sealed tubes is only a short time inevolutionary history, one is intrigued as to whether a de-tailed analysis might reveal these distinctions even in thisshort time span. The preliminary observations described inthis report also raise questions about mutation and selec-tion under the stress of limited nutrition and, perhaps,limited oxidation.

Mutation under nutrient deprivation has been of inter-est since early in bacterial genetics. In a review, Novick[14] noted that results from chemostat experiments indi-cated that starvation serves as a selective condition formutants with survival capabilities over the parental strain.

Census data exist on populations in old cultures explor-ing the concept that mutations occur despite starvationand hibernation, perhaps even in an orderly evolutionaryfashion. Using di¡erent approaches, questions of mutationin stationary phase cells and whether emerging mutantswere more ¢t than parental cells, and whether there mightbe speci¢c `survivor' mutations have been addressed. In adiscussion of di¡erences among individuals within a bac-terial clone, Koch [13] emphasized that certain genes couldbe eliminated without losing colony forming ability. `Ex-cess baggage' DNA fragments might include duplicatedgenes or fragments, fragments of ancestral genes that areno longer needed as the species evolved, and temperateviruses and insertion sequences. Also, cells in the limitedenvironment might still survive if they lost heat shock andoxidative stimulons, as well as some SOS and recombina-tion genes. Although constitutive pathways might beneeded to utilize the meager remaining nutrients, therewould be less need for induction processes. Exhaustionof some substrate molecule could lead to usage of newmetabolic pathways [15,16]. For example, Salmonella cellsin a sealed vial would no longer need genes for surfacestructures to attach to intestinal cells.

Other investigators have sought insights into broadquestions of evolution and cell biology, such as `drift ordrive' [17], `adaptive mutation' [18^24], `alive but non-cul-turable' [25], and the evolution of pathogens [26]. Amongsurvival strategies that have been described are mutationsto new metabolic pathways [16,17], and mutations in mu-tator and other repair enzyme genes [18^24]. The avail-ability of archival cultures may be a resource for answer-ing some of these questions.

4.2. Genomic loss

If it were not for the one exception, a case could bemade for the loss of the 180-kb band when genomeswere cut with AvrII in PFGE assays. Awaiting furtherexperiments and the completion of the S. typhimuriumgenome, it is uncertain whether this is a genomic loss ora genomic rearrangement (or a single base change). Sim-

ilarly, loss of some protein bands in SDS^PAGE assaysmay indicate loss of unnecessary genes for a particularenvironment. As discussed previously [27], a changefrom AUG to UUG as a start site for the rpoS genemay be an example of losing `functional baggage',although no quantitative DNA loss. The high degree ofreversion from auxotrophy to prototrophy may be a clueas to selection of mutants that utilize advantageous meta-bolic pathways. Some of the genomes examined by PFGEindeed are shorter than wild-type S. typhimurium LT2 andthere are missing bands upon Coomassie staining in SDS^PAGE assays.

4.3. Mutation of regulator genes in ¢tness selection

An intriguing concept as to adjustment to long-termstationary phase is that of a speci¢c non-null mutationin a master regulatory gene that would alter the activityof a number of genes to adjust to a paucity of resources.These mutants, named GASP (growth advantage in sta-tionary phase), are more ¢t than wild-type cells, at leasttemporarily. This concept was presented by Finkel andKolter [28] for the RpoS sigma factor and Zinser andKolter [29] for the lrp regulatory gene in Escherichia coli.We have reported that starved rpoS mutants [27,30] are ata growth disadvantage. A number of strains had altera-tions, but a majority of the isolates retained the wild-typesequence. The start site of all of these strains had UUGinstead of AUG, which would alter their translational ca-pability, but it is not known whether the original S. typhi-murium LT2 contained the UUG or AUG codon. Thischange could have occurred earlier during laboratorytransfer after isolation from a mouse.

The focus on this global regulator presents new insightsinto survival within mixed populations [31^33]. Microar-ray analysis of 4290 E. coli genes that are transcribedunder limited nutrition reveal that RpoS is pivotal in thecomplex regulation of a large fraction of total genes [34].

5. Summary

Bacterial cells remained viable after decades of storagein a sealed environment. Extensive genomic changes oc-curred, particularly with regard to changes in enzyme re-striction sites and reversion of auxotrophy to prototrophy.The genetic changes detected in these aged cultures raisequestions of necessary and dispensable genes for survivalunder the restricted environment within these sealed vials.While the observations from these experiments are intrigu-ing, the mechanisms that result in genomic and populationchanges in aged cultures have yet to be elucidated.

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Acknowledgements

Experiments were made possible by Grant ES04889-NIEHS, NIH. We thank Cheryl Nickerson for strainM3000, Mick Calcutt for experimental assistance and edi-torial review of the manuscript, and Shawna McGee formanuscript preparation.

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