Lambda Prophage Research Articles

Pan-genome analysis reveals novel chromosomal markers for multiplex PCR-based specific detection of Bacillus anthracis

BackgroundBacillus anthracis is a highly pathogenic bacterium that can cause lethal infection in animals and humans, making it a significant concern as a pathogen and biological agent. Consequently, accurate diagnosis of B. anthracis is critically important for public health. However, the identification of specific marker genes encoded in the B. anthracis chromosome is challenging due to the genetic similarity it shares with B. cereus and B. thuringiensis.MethodsThe complete genomes of B. anthracis, B. cereus, B. thuringiensis, and B. weihenstephanensis were de novo annotated with Prokka, and these annotations were used by Roary to produce the pan-genome. B. anthracis exclusive genes were identified by Perl script, and their specificity was examined by nucleotide BLAST search. A local BLAST alignment was performed to confirm the presence of the identified genes across various B. anthracis strains. Multiplex polymerase chain reactions (PCR) were established based on the identified genes.ResultThe distribution of genes among 151 whole-genome sequences exhibited three distinct major patterns, depending on the bacterial species and strains. Further comparative analysis between the three groups uncovered thirty chromosome-encoded genes exclusively present in B. anthracis strains. Of these, twenty were found in known lambda prophage regions, and ten were in previously undefined region of the chromosome. We established three distinct multiplex PCRs for the specific detection of B. anthracis by utilizing three of the identified genes, BA1698, BA5354, and BA5361.ConclusionThe study identified thirty chromosome-encoded genes specific to B.anthracis, encompassing previously described genes in known lambda prophage regions and nine newly discovered genes from an undefined gene region to the best of our knowledge. Three multiplex PCR assays offer an accurate and reliable alternative method for detecting B. anthracis. Furthermore, these genetic markers have value in anthrax vaccine development, and understanding the pathogenicity of B. anthracis.

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Exposure of Shewanella oneidensis MR-1 to Sublethal Doses of Ionizing Radiation Triggers Short-Term SOS Activation and Longer-Term Prophage Activation.

Currently, there is a lack of bacterial biomarkers indicative of exposure to ionizing radiation (IR). IR biomarkers have applications for medical treatment planning, population exposure surveillance, and IR sensitivity studies. In this study, we compared the utility of signals originating from prophages and the SOS regulon as biomarkers of IR exposure in the radiosensitive bacterium Shewanella oneidensis. Using RNA sequencing, we demonstrated that 60 min after exposure to acute doses of IR (40, 1, 0.5, and 0.25 Gy), the transcriptional activation of the SOS regulon and the lytic cycle of the T-even lysogenic prophage So Lambda are comparable. Using quantitative PCR (qPCR), we showed that 300 min after exposure to doses as low as 0.25 Gy, the fold change of transcriptional activation of the So Lambda lytic cycle surpassed that of the SOS regulon. We observed an increase in cell size (a phenotype of SOS activation) and plaque production (a phenotype of prophage maturation) 300 min after doses as low as 1 Gy. While the transcriptional responses of the SOS and So Lambda regulons have been examined in S. oneidensis after lethal IR exposures, the potential of these (and other transcriptome-wide) responses as biomarkers of sublethal levels of IR (<10 Gy) and the longer-term activity of these two regulons have not been investigated. A major finding is that after exposure to sublethal doses of IR, the most upregulated transcripts are associated with a prophage regulon and not with a DNA damage response. Our findings suggest that prophage lytic cycle genes are a promising source of biomarkers of sublethal DNA damage. IMPORTANCE The bacterial minimum threshold of sensitivity to ionizing radiation (IR) is poorly understood, which hinders our understanding of how living systems recover from the doses of IR experienced in medical, industrial, and off-world environments. Using a transcriptome-wide approach, we studied how in the highly radiosensitive bacterium S. oneidensis, genes (including the SOS regulon and the So Lambda prophage) are activated after exposure to low doses of IR. We found that 300 min after exposure to doses as low as 0.25 Gy, genes within the So Lambda regulon remained upregulated. As this is the first transcriptome-wide study of how bacteria respond to acute sublethal doses of IR, these findings serve as a benchmark for future bacterial IR sensitivity studies. This is the first work to highlight the utility of prophages as biomarkers of exposure to very low (i.e., sublethal) doses of IR and to examine the longer-term impacts of sublethal IR exposure on bacteria.

Bacterial Inactivation and Biofilm Disruption through Indigenous Prophage Activation Using Low-Intensity Cold Atmospheric Plasma.

Biofilms can be pervasive and problematic in water treatment and distribution systems but are difficult to eradicate due to hindered penetration of antimicrobial chemicals. Here, we demonstrate that indigenous prophages activated by low-intensity plasma have the potential for efficient bacterial inactivation and biofilm disruption. Specifically, low-intensity plasma treatment (i.e., 35.20 W) elevated the intracellular oxidative reactive species (ROS) levels by 184%, resulting in the activation of prophage lambda (λ) within antibiotic-resistant Escherichia coli K-12 (lambda+) [E. coli (λ+)]. The phage activation efficiency was 6.50-fold higher than the conventional mitomycin C induction. Following a cascading effect, the activated phages were released upon the lysis of E. coli (λ+), which propagated further and lysed phage-susceptible E. coli K-12 (lambda-) [E. coli (λ-)] within the biofilm. Bacterial intracellular ROS analysis and ROS scavenger tests revealed the importance of plasma-generated ROS (e.g., •OH, 1O2, and •O2-) and associated intracellular oxidative stress on prophage activation. In a mixed-species biofilm on a permeable membrane surface, our "inside-out" strategy could inactivate total bacteria by 49% and increase the membrane flux by 4.33-fold. Furthermore, the metagenomic analysis revealed that the decrease in bacterial abundance was closely associated with the increase in phage levels. As a proof-of-concept, this is the first demonstration of indigenous prophage activations by low-intensity plasma for antibiotic-resistant bacterial inactivation and biofilm eradication, which opens up a new avenue for managing associated microbial problems.

Release mechanisms and molecular interactions of Pseudomonas aeruginosa extracellular DNA.

Pseudomonas aeruginosa infection is a significant threat for clinicians. Increasing incidents of resistant biofilm infection result in high mortality rates worldwide. There is a considerable current interest in the field of extracellular DNA (eDNA)-mediated P. aeruginosa biofilm formation. eDNA acts as a glue to make biofilm more stable. This review focuses on the diverse mechanisms and factors, which enhance the eDNA release into the extracellular milieu. Furthermore, eDNA-mediated molecular interactions within the biofilm are emphasized. In addition, drug resistance mechanisms due to the versatility of eDNA are discussed. Spatial physiological diversity is expected due to different metabolic activity of bacterial subpopulation present in P. aeruginosa biofilm layers. In P. aeruginosa, eDNA release is accomplished by cell lysis and OMVs (outer membrane vesicles). eDNA release is a spontaneous and multifactorial process, which may be accomplished by PQS, pyocyanin, and lambda prophage induction. Hydrogen peroxide and pyocin trigger cell death, which may facilitate eDNA release. Lung mucosa of cystic fibrosis patients is enriched with eDNA, which acidifies biofilm and develops P. aeruginosa resistance to aminoglycosides. Further studies on spatial and molecular characterization of bacterial subpopulation in biofilm will shed light on eDNA-biofilm interaction more precisely.Key Points• Extracellular DNA (eDNA) is a key component of Pseudomonas aeruginosa biofilm.• P. aeruginosa eDNA acts as a glue to make biofilm more stronger.• Bacterial cell death or lysis may be the potential way to release P. aeruginosa eDNA into extracellular milieu.• P. aeruginosa eDNA contributes to develop resistance to antimicrobials.

Simple and efficient genome recombineering using kil counter-selection in Escherichia coli

Seamless modification of the Escherichia coli genome using positive selection / negative selection is widely used in metabolic engineering and functional genome analysis. Some excellent negative selection systems have been reported, of which tetA-sacB and inducible toxins system are prominent. To expand the existing negative selection toolkit, we constructed a new negative selection marker system based on kil gene of lambda prophage. The selection stringency of kil was measured and compared with the most widely used counter-selection gene, sacB, at the lacI, ack, and dbpa loci using different E. coli strains. At all these loci of tested strains, the selection stringency of kil significantly exceeds that of sacB by 2- to 28-fold. When dsDNA fragments were employed for recombination, the efficiency for isolating the correct recombinant of kil was significantly higher than that of sacB. This new negative selection system does not require special media or extended incubation time. However, our system cannot be used in host strains containing temperature-sensitive kil gene. A Red system providing plasmid without kil gene is recommended for use together with our system. Our counter-selection system is expected to be an addition to the engineering arsenal of E. coli.

An enhanced bioindicator for calorimetric monitoring of prophage-activating chemicals in the trace concentration range.

Viruses that infect bacteria (bacteriophages) can either lyse bacteria directly or integrate their genome into the bacterial genome. In the latter case, the viral genome (called prophage) remains dormant, and both phages and bacteria are able to survive in this state. But the silent prophages can be reactivated by, e.g., chemicals, accompanied by the release of substantial quantities of phage particles that further infect other phage-sensitive bacteria, thus harming ecosystems or technical systems by way of a viral bloom. Recently, a calorimetric method was developed to monitor the prophage-activating properties of chemicals. The method evaluates the difference in the metabolic heat of the Escherichia coli bioindicator with (λ+) and without (λ-) lambda prophages under the influence of the test substances. Simulations and experiments clearly demonstrate that the sensitivity of the test can be significantly improved, when a customized mixture of λ+ and λ- E. coli strains is used for enhanced bioindication. Hence, the new method mirrors a common situation in nature, where bacteria with and without prophages coexist. In summary, a monitoring method is suggested that provides quick results (after few hours) and offers both the option for automation with low workload (requires only a few minutes) and usage of commercially available instruments.

Rapid Identification of E. coli Bacteriophages using Mass Spectrometry

Objective: The current increasing interest in the application of mass spectrometry, in particular MALDI-TOF MS, to identification of bacteria and fungi calls for a need to utilise this technology for identification of other infectious agents such as viruses. The aim of the present study was to develop a rapid and reliable mass spectrometry-based proteomic method for identification of Escherichia coli phages. Methods: The approach was based on rapid in-solution tryptic digestion of suspensions of plaque-purified bacteriophage followed by mass spectral analysis. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography – tandem mass spectrometry (LC-MS) were used to analyse the tryptic digests. Processing of tandem mass spectrometry data and interpretation of results were achieved using Mascot software and the Swiss-Prot database. Results: Five bacteriophage species (Enterobacteria phages P2, T4, T5, T7 and Lambda) isolated in E. coli cultures were identified. The viral proteins were identified from a complex mixture of host bacterial proteins. In addition, using a single ion monitoring method, a Lambda prophage derived protein was also identified. Conclusion: The data obtained demonstrate that LC-MS/MS can be used for accurate identification of E.coli- specific bacteriophages in both lytic and lysogenic cycles

In Search of an Explanation

In Search of an Explanation

The Structural Characteristics and Function Analysis of the Mobile Genomic Islands Flanked by Isocitrate Dehydrogenase Genes in &lt;i&gt;Escherichia coli&lt;/i&gt; and &lt;i&gt;Salmonella enterica&lt;/i&gt;

Eleven genomic islands (GIs) flanked by isocitrate dehydrogenase genes are determined in Escherichia coli and Salmonella enterica. These GIs have at least one mobile gene, such as integrase gene, transposase gene or recombinase gene. Through annotation of internal genes, these GIs are related to lambda prophage. The excisionase gene is associated with the mobile gene in some GIs. An ABC transporter, namely, sitABCD operon, is existed in some GIs and may uptake Fe2+ and Mn2+. Mn2+ is a second cofactor and an essential activator of the isocitrate dehydrogenase. The cleavage site of functional lambda integrase is 5’-TGCTGCGCCA-3’ in direct repeats at 3’-end of icd gene.The truncated lambda integrases (ECP_1132 and ECP_1135) are inactive because the transposon inserted the integrase gene by 5’-CCTGG-3’. This Fe2+/Mn2+ transport operon is predicted that is a recent product of horizontal gene transfer in E. coli because this operon is also existed in S. enterica and is not in a mobile GIs.

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host's chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.

Influence of the Escherichia coli oxyR gene function on λ prophage maintenance

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of λ prophage. Here, we demonstrate that H2O2-mediated λ prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a λ lysogen in the presence of H2O2. On the other hand, stimulation of the p M promoter by cI857 overproduced from a multicopy plasmid was decreased in the ΔoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p M promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p M promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced λ prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating λ prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.

Calorimetric real time monitoring of lambda prophage induction

Calorimetry can be used for a noninvasive monitoring of metabolic and energy changes in bacterial cultures in real time. This potentially includes changes occurring during phage infection cycles. To demonstrate this, the growth of Escherichia coli K124 F − carrying the lambda prophage was monitored in a reaction calorimeter, while inducing the shift from lysogeny to lysis using methotrexate (MTX) as the inducer. Different phases of the phage infection were distinguishable calorimetrically. The calorimetric signals corresponded well to changes in growth stoichiometry and kinetics that were measured non-calorimetrically. The comparison of the calorimetric results with other measurements of activity revealed that the calorimetric method was superior since the calorimetric data mirrored the progress of infection in more detail. Contradictions between the calorimetric data and the appearance of infectious phages could result from inactivation of phage by MTX.

AFM Studies of DNA Loops Secured by Lambda Repressor Proteins

Large, cooperative assemblies of proteins that wrap and/or loop genomic DNA may shift configurational equilibria that determine developmental pathways. Such is the case of the lambda bacteriophage which may exhibit lytic or lysogenic growth. The lysogenic state of lambda prophages is maintained by the lambda repressor (CI), which binds to tripartite operator sites in each of the OL and OR control regions located about 2.3 kbp apart on the phage DNA and represses lytic promoters. Dodd and collaborators have suggested that an initial loop formed by interaction between CI bound at OR and OL provides the proper scaffold for additional CI binding to attenuate the PRM promoter and avoid over production of CI. The looping equilibrium as a function of CI concentration has been measured using tethered particle motion analysis, but the oligomerization of CI in looped states could not be determined. Scanning force microscopy has now been used to probe this directly. An equilibrium distribution of looped and unlooped molecules on planar poly-L-ornithine-coated mica was found to be commensurate to that for tethered molecules in solution, and the occupancies of specific operator sites for several looped and unlooped conformations were determined. Some loops appeared to be sealed by oligomers of 6-8, most by oligomers of 10-12, and a few by oligomers of 14-16.

AFM Studies of &amp;#955; Repressor Oligomers Securing DNA Loops

Large, cooperative assemblies of proteins that wrap and/or loop genomic DNA may "epigenetically" shift configurational equilibria that determine developmental pathways. Such is the case of the lambda bacteriophage which may exhibit virulent (lytic) or quiescent (lysogenic) growth. The lysogenic state of lambda prophages is maintained by the lambda repressor (CI), which binds to tripartite operator sites in each of the O(L) and O(R) control regions located about 2.3 kbp apart on the phage genome and represses lytic promoters. Dodd and collaborators have suggested that an initial loop formed by interaction between CI bound at O(R) and O(L) provides the proper scaffold for additional CI binding to attenuate the P(RM) promoter and avoid over production of CI. Recently, the looping equilibrium as a function of CI concentration was measured using tethered particle motion analysis, but the oligomerization of CI in looped states could not be determined. Scanning force microscopy has now been used to probe these details directly. An equilibrium distribution of looped and unlooped molecules confined to a plane was found to be commensurate to that for tethered molecules in solution, and the occupancies of specific operator sites for several looped and unlooped conformations were determined. Some loops appeared to be sealed by oligomers of 6-8, most by oligomers of 10-12, and a few by oligomers of 14-16.

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage λ and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.

Marking embryonic stem cells with a 2A self-cleaving peptide: a NKX2-5 emerald GFP BAC reporter.

BackgroundFluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.MethodologyHere, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice.ConclusionsOur results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library.

Cloning and expression of hepatitis C virus core protein in pGemex-1 expression vector.

Hepatitis C virus is one of the main causes of chronic hepatitis in developing countries. There are 170 million affected people around the world as reported by the World Health Organization. The treatment of hepatitis C is not successful in most cases; it is extremely costly, and requires prolonged therapy, therefore it is desirable to develop a vaccine to prevent the spread of hepatitis C virus. Hepatitis C virus RNA was extracted from a hepatitis C virus-infected serum sample. cDNA was synthesized and the hepatitis C virus core gene was amplified by polymerase chain reaction. The polymerase chain reaction product was cloned in pGEMEX-1 expression vector and expressed in Escherichia coli BL21 strain with DE3 (a lambda prophage carrying the T7 RNA polymerase gene) host by induction of promoter using one millimolar isopropyl beta-D-thiogalactopyranoside in laboratory scale. Induced lysate cells were electrophoresed on SDS- polyacrylamide gel. A protein band was detected in induced cells in comparison with non-induced cells. Expressed protein was confirmed by gel diffusion and dot blot analysis using induced lysate cells as antigen and hepatitis C virus-infected serum as antibody. In present study, we have provided a recombinant plasmid based on hepatitis C virus core gene.

Host responses influence on the induction of lambda prophage

Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.

Phage Integration and Chromosome Structure. A Personal History

In 1962, I proposed a model for integration of lambda prophage into the bacterial chromosome. The model postulated two steps (i) circularization of the linear DNA molecule that had been injected into the cell from the phage particle; (ii) reciprocal recombination between phage and bacterial DNA at specific sites on both partners. This resulted in a cyclic permutation of gene order going from phage to prophage. This contrasted with integration models current at the time, which postulated that the prophage was not inserted into the continuity of the chromosome but rather laterally attached or synapsed with it. This chapter summarizes some of the steps leading up to the model including especially the genetic characterization of specialized transducing phages (lambdagal) by recombinational rescue of conditionally lethal mutations. The serendipitous discovery of the conditional lethals is also described.