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Bacterial Mutation

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The Bacterial Reverse Mutation Test

Annie Hamel, ... Ray Proudlock, in Genetic Toxicology Testing, 2016

Abstract

Bacterial mutation tests using various Salmonella typhimurium LT2 and Escherichia coli WP2 strains are by far the most widely used systems for predicting mutagenicity of chemicals and other materials including complex mixtures and environmental contaminants. The tests are generally required worldwide by regulatory authorities as an initial screen for potential long-term adverse health effects of new materials and chemicals, including drugs, agrochemicals, medical devices, and household agents. The initial sections of this chapter describe the theoretical background and the history of the test to allow a better understanding of the principles of its performance and interpretation. Rigorous standardized procedures necessary to perform tests suitable for screening and regulatory submission are presented that closely follow the recommendations of the developers of the test and regulatory guidelines and are based on the scientific community's 40 years of experience with the test. All aspects of routine testing including bacterial strain purification, maintenance and characterization, use of S9 mix, concurrent controls, handling and interpretation of results, as well as clarification of unexpected findings or borderline results are covered. These procedures should give reproducible results in the shortest timeframe and minimize workload while avoiding problems, errors, and repeat testing. Variations of the test including screening versions used at an early stage of development, when test article supply and resources are limited, are discussed briefly.

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Evolutionary Mechanisms and Diversity in Cancer

Henry H.Q. Heng, ... Christine J. Ye, in Advances in Cancer Research, 2011

A Linear Evolutionary Models

Evolutionary studies of bacteria mutations (Brash and Cairns, 2009; Cairns, 1975), hematological malignancies (Nowell, 1976; Rowley, 1988), and the conventional ideas of evolution that the accumulation of small alterations leads to big changes, have influenced the generally accepted idea that the evolutionary process of tumorigenesis occurs through multiple linear steps (Hanahan and Weinberg, 2000; Huang et al., 2009; Nowak et al., 2006; Nowell, 1976; Vogelstein and Kinzler, 2004). In such a linear model, the evolutionary process of cancer has been thought to be mainly based on the accumulation of gene mutations with chromosome change playing a primarily ancillary role. In many types of hematological cancers where commonly shared marker chromosomes can be identified, focus is placed on the unique pattern of the abnormal chromosome itself, assuming that the rest of the genome is unaffected, and other changes are insignificant as they are more or less random. According to the prevailing theory, a stepwise pattern based on gene trees can be identified during cancer evolution and key gene mutations driving cancer evolution can be identified and targeted for medical intervention. There are some very exciting studies of solid tumors that demonstrate the power of evolutionary analysis in this manner (Maley et al., 2006). Interestingly, most successful stories are linked to slowly progressing types of cancer. In most cancers it is difficult to illustrate the main pathway of evolution, as there are too many genome-level alterations and large numbers of gene mutations that make building a gene tree difficult. The fact that every tumor would have a different tree limits the impact of this type of study. Increasing data show that the dynamics of cancer evolution is much higher than that of natural evolution (Heng, 2007b; Heng et al., 2010a), as many conserved genetic elements are shared among different species, yet are drastically different among cancer samples (Calin et al., 2007).

Mitelman and colleagues have systematically analyzed karyotype evolution in various types of cancers with a focus on patterns of clonal evolution. Through this they have established relationships between cytogenetic subgroups and karyotypic pathways. However, of the 50,000 tumor cases that have been reported, there are over 40,000 karyotypic aberrations suggesting that no commonly shared, recurrent patterns exist in the majority of cases, particularly in solid tumors (Mitelman, 2006). Even though clonal evolution can be observed in some cases during different stages of primary tumors and metastatic cancers, and diverse karyotypes can be grouped according to their statistical behavior (Höglund et al., 2005), the presence of karyotypic heterogeneity is underreported and overwhelming. Chromosomal instability has been blamed for this high heterogeneity (Gisselsson, 2003; Heng et al., 2004). Understanding the consequences of karyotypic alterations to the overall system and the meaning of the long ignored nonclonal chromosomal aberrations (NCCAs) is an additional challenge, as now we know that population heterogeneity can change the genetic landscape, affecting evolution.

Another important topic is the mathematical and computational modeling of somatic cell evolution in tumorigenesis. Various factors have been studied by Gatenby's group including the competition between normal and cancer tissue, the contributions of microenvironments, and the dynamics of the evolutionary landscape (Gatenby, 1996; Gatenby et al., 2010; Vincent and Gatenby, 2008). In addition to using agent-based computational models to illustrate the promising strategies of benign cell boosters and chemosensitive boosters (Maley et al., 2004), a number of key features of cellular alteration (self-sufficiency in growth signals; insensitivity to antigrowth signals; evasion of apoptosis; limitless replicative potential; sustained angiogenesis and tissue invasion and genetic instability) or hallmarks of cancer have been analyzed in a three-dimensional stochastic multistep model (Spencer et al., 2006). The predictions regarding genetic instability are particularly interesting. By illustrating that early-onset and late-onset tumors take different mutational paths to cancer, and that genetic instability is often the cause of mutations in earlier onset tumors, this type of analysis demonstrates the mechanism of evolutionary dynamics. Similarly, simulations have been used to study tumor growth using the hybrid discrete continuum model (Anderson et al., 2006). The colorectal tumor evolution process (waiting time and transition from benign, invasive and metastatic phase) has been analyzed based on common mutations (Beerenwinkel et al., 2007; Jones et al., 2008). However, most of these models are based on the gene theory while the key contributions of the genome have been ignored. For example, many of these approaches have considered carcinogenesis as a micro-evolutionary process where multiple genetic and epigenetic changes accumulate in a single cell (Foo et al., 2011; Tarafa et al., 2008).

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Nanotoxicology: evaluating toxicity potential of drug-nanoparticles

Vandana Patravale, ... Ratnesh Jain, in Nanoparticulate Drug Delivery, 2012

Bacterial mutation assay (Ames test)

The bacterial mutation assays offer quick and uncomplicated evaluation of carcinogenic or mutagenic effects by detecting alterations in normal phenotype of organisms or detecting the reversion to normal phenotype. The former method, referred to as the forward mutation assay, detects the nucleotides which upon alteration lead to loss of expression or function of a gene; however, the method is practically less reliable. On the other hand, the latter method, also known as the backward mutation assay, assesses a few nucleotides which upon modification restore normal functioning of a defective gene [35,37,38]. With regards to microbial mutation assays, S. typhimurium and E. coli constitute two of the sensitive, well-known and validated systems for assessing primarily three kinds of mutations viz. the frameshift mutation affecting the reading frame of DNA due to insertion or deletion of one/few base pairs, the base-pair substitution mutations affecting the DNA structure due to replacement of an inherent base with an alternative one and DNA cross-linking connecting the two DNA strands [39].

The microbial strains used in these assays exhibit malfunctioning of one of their functional genes such as a defect in genes associated with histidine and tryptophan synthesis necessitating their addition to the growth medium in absence of a mutational reversion to the normal prototype. Yet other modifications have been introduced for an increased sensitivity such as a mutation in uvrA gene of E. coli and in uvrB gene of S. typhimurium affecting their DNA repair mechanism upon UV exposure or incorporation of plasmid pKM101 affording ampicillin resistance or rfa wall mutation in S. Typhimurium increasing their cell wall permeability to large molecules and affording an increased sensitivity to crystal violet. Effective detection of mutagenic potential is usually conducted employing a battery of several tester bacterial strains since each one is capable of detecting only a single form of damage. S. typhimurium strains TA98, TA1537, TA1538, TA97 and TA97a are employed to detect reversion from his–to his+ due to frameshift mutations while TA100 and TA1535 are converted to histidine prototrophy by base substitution mutations. TA102 is sensitive for detecting oxidative mutations and DNA cross-linking [40,41]. The E. coli strains identify conversion to tryptophan prototrophy due to base-pair substitution mutations [42]. The regulatory agencies normally recommend a battery of strains; the ones recommended by OECD and ICH include one strain from: (1) TA98; (2) TA100; (3) TA1535; (4) TA1537, TA97 or TA97a; (5) TA102, WP2 uvrA or WP2 uvrA (pKM101 and for detecting crosslinking: TA102 or to add WP2 (pKM101) [43]. Well-characterized, appropriately documented and reliable tester strains may be obtained from a number of recognized suppliers which include MolTox (Boone, North Carolina, USA) and Invitrogen (Rockville, Maryland, USA) while E. coli strains are supplied by National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland, and from MolTox [40].

ICH approves two approaches of conducting bacterial mutation assays:

Two-phase approach where a preliminary toxicity assay (first phase) determines the dose range for a particular assay while the mutagenicity assay (second phase) confirms the DNA damaging potential. This approach is common in the United States and Europe.

Countries like Japan follow the approach of performing a preliminary experiment, the toxicity-mutation assay, employing all the strains with concomitant positive and negative controls and testing on duplicate plates instead of one or two representative strains and single plate per dose [44].

Many test substances are not direct inducers of mutagenicity but are triggered by the mammalian activation systems involving nicotinamide adenine dinucleotide phosphate (NADPH), molecular oxygen-or cytochrome P450-dependent mixed-function oxygenases. As the aforementioned test bacteria lack these metabolic activities external activation is conducted employing established protocols and chemical compounds [35].

Functional accuracy of the test system is ascertained by simultaneously maintaining appropriate controls. Functioning of tester strains is controlled by maintaining negative controls for tester strain-activation combination. The vehicle used to suspend the nanoparticles serves as the negative control while known carcinogens/mutagens or compounds requiring activation by the exogenous activators serve as the positive controls. Examples of the latter include 2-Aminoanthracene requiring exogenous activation for the strains TA98, TA100, TA1535, TA1537, TA1538, Mitomycin C for TA102, Methyl methanesulfonate for E. coli, etc. A list of other positive controls has been published in reference [35] of this chapter.

At the completion of the assay the bacterial background lawn is carefully evaluated [35] to omit the nonrevertant background lawn colonies from the test scores. The results of the test formulations are compared with those of the vehicle control. To confirm the genotype of any dubious colonies, they are transferred to the specific amino acid-devoid medium and only the organisms exhibiting growth in such mediums are scored as true revertants. Results are presented as total number of revertants per plate. A compound is considered to be toxic in the event of greater than 50% reduction in the mean number of revertants when compared to the vehicle control value or moderate reduction in background lawn [35]. The test outcome is regarded as positive in the event of dose-dependent increase in number of revertants per plate, in presence or absence of exogenous activator, with values at the peak of the dose response being greater than or equal to twice or thrice the vehicle control value. Outcomes are regarded as equivocal in case of a dose-dependent increase in mean revertant value that does not reach this threshold or a non dose-dependent increase that equals or crosses the threshold. A non-positive or non-equivocal response is regarded as negative. ICH and OECD require retesting of equivocal outcomes using alternative doses, exogenous activators and treatment methods [43,44].

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Boron

Sinan Ince, ... Begum Yurdakok-Dikmen, in Reproductive and Developmental Toxicology (Second Edition), 2017

Mutagenicity/Genotoxicity

According to in vitro mutagenicity studies, involving bacterial mutation assays in Salmonella typhimurium and Escherichia coli, the mutagenic activity of boric acid, borax, or disodium octaborate tetrahydrate was not observed in bacterial DNA damage assay, unscheduled DNA synthesis, chromosomal aberration, and sister chromatid exchange in mammalian cells (Arslan et al., 2008; Duydu et al., 2015; Hubbard, 1998; National Toxicology Program, 1987; Selier, 1989). Inorganic borates have also been reported not to be mutagenic in vivo (EPA, 2004). When boric acid was administered orally in deionized water to Swiss Webster mice for 2 days at 900, 1800, or 3500 mg/kg doses, no induction of chromosomal aberrations or mitotic spindle abnormalities was found in bone marrow erythrocytes (EFSA, 2004).

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Methylcholanthrene, 3-

H Robles, in Encyclopedia of Toxicology (Third Edition), 2014

Genotoxicity

3-MC has been found to cause bacterial mutations, chromosome breaks in bone marrow, and induce DNA adducts in animals. 3-MC was also found to form DNA adducts and mutations. Increased numbers of chromosomal aberrations have been reported to be a sensitive marker for exposure to PAHs.

3-MC is mutagenic in a number of in vitro and in vivo assays and is used regularly as a positive control in these assays. 3-MC has been shown to covalently bind to DNA and other macromolecules. Using TA100 strain of Salmonella in the Ames test, S-9 liver microsomal fractions from mice, rats, hamsters, pig, and humans produced a doubling of the reversion rate for all liver fractions except the pig. 3-MC has also produced positive findings in the Ames test using S-9 rat liver microsomal fractions stimulated with Aroclor-1254 in strains TA100, TA1535, TA1537, TA1538, and in TA1538, TA98 with rat liver S-9 fractions stimulated with phenobarbital. Positive mutagenic findings were also noted in the Chinese hamster V-79 thioguanine assay, in unscheduled DNA assays done in human fibroblasts, in rodent primary cells and hepatocytes, in the Chinese hamster ovary hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay but not in several Escherichia coli WP2 uvrA assays with S-9 activation. Positive findings were also reported in human lymphocyte assays showing increased sister chromatid exchanges.

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History of Microbiology

W.C. Summers, in Encyclopedia of Microbiology (Third Edition), 2009

Bacterial Transformation and DNA

With the beginning of the understanding of the process of bacterial mutation as well as the discovery of ways of manipulation of bacterial genes by conjugation, the field of microbial genetics entered the modern period. Two disparate strands of research led to the current understanding of the nature of genes in bacteria. One strand comes from work on bacterial virulence and pathogenesis in pneumonia, and led to the identification of DNA and the chemical stuff of which genes are composed. The other strand comes from the study of bacteriophage replication.

In 1928, Frederick Griffith (1877–1941) was investigating the virulence of the organism that is responsible for pneumonia (Streptococcus pneumoniae or 'pneumococcus'), and he found that heat-killed bacteria of one form of pneumococcus could somehow convert live bacteria of another form to exhibit some of the antigenic and virulence properties of the heat-killed form. While these results were of no apparent interest or relevance to the few bacteriologists interested in microbial heredity, they were of real importance to the pathologists interested in pneumonia. Oswald T. Avery (1877–1955), working at the Rockefeller Institute, was one of several scientists who confirmed and followed up Griffith's work with a research program on what he called 'transformation' of antigenic types. Work in his laboratory, first by Martin Dawson followed by James L. Alloway, and then by Colin MacLeod (1909–72) and Maclyn McCarty (1911–2005), eventually led to characterization of the transforming material from the heat-killed bacteria as DNA itself. The final characterization, published in 1944, relied on the use of the newly purified and characterized enzymes, DNase and RNase, as well as several well-known proteolytic digestive enzymes. Still, appreciation of DNA was not universal: at the mid-century meeting of the Genetics Society of America, DNA was hardly mentioned.

Because proteins exhibited the diversity expected of genes and the chemistry of DNA as it was then understood suggested its 'information content' was too low to have genetic potential, DNA was not taken seriously as the genetic material. Two major works soon challenged that belief: using new analytical methods resulting from wartime research, Rollin D. Hotchkiss (1911–2004) showed that the base compositions of DNAs from different organism were not identical and Erwin Chargaff (1905–2002) noted certain regularities in the analyses of all DNAs: the number of purines always equal the number of pyrimidines, and the ratios of adenine to thymine and guanine to cytosine were always very near to one.

While the chemistry of the 'transforming principle' in pneumococcus was being established, a second line of work was going on to understand the process of gene duplication. Max Delbrück saw bacterial viruses (bacteriophage) as a simple model for the process of gene replication and he recruited a group of like-minded associates to attack this problem. In the early stages of their work, the American Phage Group (as Gunther Stent has named Delbrück's school) treated the host bacterium more or less as a 'black box' and studied phage replication as a simple input–output process. In a widely cited experiment, in 1952 Alfred Hershey (1908–97) and Martha Chase (1927–2003) labeled bacteriophage with two isotopes, 32P in the DNA, and 35S in the protein, and in an attempt to follow the fate of the two major components of the phage through one life cycle, they noted that most of the 32P label entered the cell and a significant fraction ended up in progeny phage, while very little of the 35S label entered the cell and even less ended up in the progeny phage. While this result is often described in texts and reviews as if the results were 'all or none', the experimental results given in the original paper, while certainly supportive of the 'only DNA' hypothesis, are far from conclusive.

Another member of the American Phage Group was James D. Watson (1928–), a student of Luria in Indiana University. Watson's thesis was on the radiobiology of bacteriophage and he firmly believed that DNA was the chemical substance of the gene. Watson and Francis Crick (1916–2004), working at the Cavendish Laboratory in Cambridge England, devised a plausible model for the three-dimensional structure of DNA, which finally provided the much-needed explanatory framework for the genetic role of DNA (1952). Accounts of this landmark research abound, and do not need repetition here.

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Alternative In Vitro Models for Safety and Toxicity Evaluation of Nutraceuticals

Gopala Krishna, Gopa Gopalakrishnan, in Nutraceuticals, 2016

Mammalian Cell Mutation Assays

These assays are the mammalian counterparts to the "Ames" bacterial mutation tests. Because these assays are conducted with mammalian cells that have a much longer cell division time (12–24 h compared to 20–30 min for bacteria), they require a proportionately longer assay time compared to bacterial tests, generally 4–8 weeks. Furthermore, because culture times are greater, the risks for culture contamination and subsequent repeat testing are also increased. Although there are several different mammalian systems that are used for assessing induction of point mutations in mammalian cells, the current systems of choice are the Chinese hamster ovary (CHO)/HGPRT assay system and mouse lymphoma assay system.

Chinese hamster ovary (CHO)/HGPRT assay system: The HGPRT (hypoxanthine guanine phosphoribosyl transferase) gene is used as the basis for mutant selection for detecting forward mutations (normal to mutant). This gene is located on the X-chromosome that is functionally hemizygous in ovary cells (female). Therefore, only one chromosome (gene copy) needs be mutated to observe the mutant phenotype (gene expression). In normal cells, the HGPRT gene is used for purine salvage for the formation of ATP and GTP. In the absence of a functional HGPRT gene, the cell can still produce these nucleotides de novo. Therefore, a mutation at this locus alone is generally not lethal. As a means for selecting mutant cells, cells are cultured in the presence of the antimetabolite 6-thioguanine (6-TG). A normal "wild-type" cell with a functional HGPRT gene will incorporate the toxic antimetabolite in place of guanine, resulting in the death of the normal cell. Conversely, a cell with a mutant HGPRT gene will not take up the antimetabolite and will survive through de novo synthesis of purines.

Because mutagenicity is typically correlated with cytotoxicity, it is necessary to perform preliminary cytotoxicity tests to fully assess the dose-response curve prior to dose selection. To assess induction of mammalian mutations, duplicate cultures of CHO cells are cultured for 4 h with several concentrations of xenobiotic in the presence or absence of metabolic activation. After treatment, known numbers of cells are cloned to determine nutraceutical-induced cytotoxicity, whereas a subset of cells is carried in culture for 7–9 days to express the mutant phenotype (clear out endogenous levels of hypoxanthine and guanine). Mutants are then selected by culturing a million cells at each concentration in the presence of the antimetabolite (6-TG). After an additional week in culture, each mutant cell will form a colony that is visible to the naked eye.

Data interpretation: Increased numbers of mutant colonies per million cells compared to spontaneous numbers of mutants in the control cultures are indicative of a "positive" induction of mutations. This assay is less sensitive than the bacterial mutation assays because it is conducted with a mammalian cell line that has some mechanisms for DNA repair and because mammalian DNA is afforded some protection from chromosomal proteins that are not present in bacteria. In addition, this test is not sensitive to larger frameshift mutations and chromosomal aberrations that usually affect other essential (needed for survival) genes downstream from the HGPRT gene. Therefore, the absence of mammalian gene mutations in this assay does not negate or reduce risk if effects are seen in other assays. Conversely, a positive response in this assay in addition to a positive response in one or more other in vitro assays has a high risk for further product development.

Mouse lymphoma assay (MLA): MLA can detect a variety of mutations, including point mutations, and is performed similar to HGPRT assay. In contrast to the in vitro micronucleus test (described in this chapter), MLA also detects translocations (Liechty et al., 1998). Most of the substances that are positive in this mammalian gene mutation test also induce clastogenic effects (Kirkland et al., 2011).

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The In Vitro Chromosome Aberration Test

Marilyn Registre, Ray Proudlock, in Genetic Toxicology Testing, 2016

7.6.15 S9 Mix

S9 mix may be prepared in the same way as for the bacterial mutation test, although some laboratories use different buffers/diluents. The concentration of S9 fraction in the S9 mix depends on the laboratory and test system. We suggest 15% and 10% v/v for CHO and HPBL, respectively, which both yield a final concentration of 2% v/v after dilution in culture medium. Typically, the final concentration of S9 fraction in the culture medium is 1–2%; higher concentrations may inhibit cell growth. In addition, S9 mix contains the following cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate, and 4 mM NADP [40]; therefore, each 1 mL of S9 mix contains:

10%15%Water0.335 mL0.285 mLPhosphate buffer 0.2 M pH 7.40.500 mL0.500 mLNADP 0.1 M0.040 mL0.040 mLG6P0.005 mL0.005 mLKMg0.020 mL0.020 mLS9 fraction0.100 mL0.150 mL

All components should be sterile and added aseptically in the proportions and order listed here to a sterile container on ice, kept on ice or refrigerated, and used on the day of preparation.

Unused S9 mix should be discarded and not frozen for future use because it rapidly loses activity.

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Evolution, Theory and Experiments with Microorganisms

R.E. Lenski, M.J. Wiser, in Encyclopedia of Microbiology (Third Edition), 2009

Replica plating experiment

Joshua and Esther Lederberg devised a more direct demonstration of the random origin of bacterial mutations, which they published in 1952. In their experiment, thousands of cells are spread on an agar plate that does not contain the selective agent; each cell grows until it makes a small colony, and the many colonies together form a lawn of bacteria (master plate). By making an impression of this plate using a pad of velvet, cells from all of the colonies are then transferred to several other agar plates (replica plates) that contain the selective agent, which prevents the growth of colonies except from those cells with the appropriate mutation. If mutations are caused by exposure to the selective agent, then there should be no tendency for mutant colonies found on the replica plates to be derived from the same subset of colonies on the master plate. However, if mutations occur during the growth of the colony on the master plate (i.e., prior to exposure to the selective agent), then those master colonies that give rise to mutant colonies on one replica plate should also give rise to mutant colonies on the other replica plates. Indeed, Lederberg and Lederberg observed that master colonies giving rise to mutants on one replica plate gave rise to mutants on the other replica plates. Moreover, they could isolate resistant mutants from those master colonies, without the cells having ever been exposed to the selective agent. This experiment thus demonstrates that the mutations had occurred randomly during the growth of the colony on the master plate.

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Boric Acid and Inorganic Borate Pesticides

Craig E. Bernard, ... Mark J. Manning, in Hayes' Handbook of Pesticide Toxicology (Third Edition), 2010

(a) Genotoxicity

A number of in vitro mutagenicity studies have been conducted with boric acid, disodium tetraborate decahydrate, or disodium octaborate tetrahydrate, including bacterial mutation assays in Salmonella typhimurium and Escherichia coli, gene mutation in mammalian cells (L5178Y mouse lymphoma, V79 Chinese hamster cells, C3H/10T1/2 cells), bacterial DNA-damage assay, unscheduled DNA synthesis (hepatocytes), chromosomal aberration and sister chromatid exchange in mammalian cell (Chinese hamster ovary, CHO cells). No evidence of mutagenic activity was observed (Bakke, 1991; Haworth et al., 1983; Landolph, 1985; NTP, 1987; Stewart, 1991). In addition, no mutagenic activity was seen in vivo in a mouse bone marrow micronucleus study on boric acid (O'Loughlin, 1991).

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Bacterium Mutant

Bacterial mutants have often lost some growth property (e.g., failure to utilize a particular carbon or nitrogen source or failure to grow without a particular nutrient), or acquisition of some new growth property (e.g., ability to grow in the presence of some toxic substance).

From: Encyclopedia of Biodiversity (Second Edition), 2013

Related terms:

Wild Type

Antibiotics

Nested Gene

Bacterium

Mutation

Bacteriophage

Pseudomonas aeruginosa

Escherichia coli

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Bacterial Genetics

Stanley Maloy, in Encyclopedia of Biodiversity (Second Edition), 2013

Effects of Mutations

Bacterial mutants are typically described by comparison to a standard, well-characterized, reference strain called the 'wild-type' strain. Bacterial mutants have often lost some growth property (e.g., failure to utilize a particular carbon or nitrogen source or failure to grow without a particular nutrient), or acquisition of some new growth property (e.g., ability to grow in the presence of some toxic substance). Genes can be divided into two categories based on the phenotypes of the corresponding mutants: nonessential gene products are only required under specific growth conditions, whereas essential gene products are required under all conditions. The genes of lactose catabolism are nonessential because they are only required for growth on medium with lactose as the sole carbon source. In contrast, the genes encoding RNA polymerase are essential because they are required for growth on all media. Null mutations in a nonessential gene will prevent growth on a medium that requires that gene product but such mutants will still grow on other media. In contrast, null mutations in an essential gene are lethal. Consequently, such mutants cannot be recovered from haploid bacteria. Nevertheless, it is possible to isolate more subtle mutations in essential genes. For example, it is possible to isolate mutations that alter a subunit of RNA polymerase that make the organism resistant to the antibiotic rifampicin. It is also possible to isolate mutations where some phenotype is observed under certain 'nonpermissive' conditions but not under other 'permissive' conditions (Table 1).

Table 1. Some types of conditional mutations used in bacterial genetics

Conditional mutationPermissive conditionsNonpermissive conditionsTemperature sensitive (Ts)30 °C42 °CCold sensitive (Cs)42 °C30 °COsmoremedialHigh osmotic strengthLow osmotic strengthSuppressor sensitiveHost with suppressor mutationHost lacking suppressor mutation

Because not all mutations have an observable effect, it is important to distinguish the genotype from the resulting phenotype. In bacterial genetic nomenclature a three-letter mnemonic refers to a pathway or discrete cluster of physiologically connected systems. A fourth, capitalized letter represents a particular gene of that set. The genotype is written in lower case letters and italicized (e.g., purB), with a plus superscript indicating the wild-type genotype. (The purB gene encodes one of the enzymes required for purine biosynthesis.) The phenotype is indicated by the same mnemonic but the first letter is upper case and it not italicized (e.g., PurB), with a plus superscript indicating the functional phenotype and a minus indicating a mutant phenotype. The genotype of a cell is usually inferred from its phenotype but may also be determined indirectly by recombination experiments or directly by DNA sequencing

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Research on Nitrification and Related Processes, Part A

Luis A. Sayavedra-Soto, Lisa Y. Stein, in Methods in Enzymology, 2011

6 Conclusions

The production of an AOB mutant may require a 2–6-month investment; however, the possible outcomes can be highly valuable. AOB are important players in the nitrogen cycle initiating the oxidation of ammonia and in generating traces of the NO and N2O gases. Through mutagenesis, it was possible to research the roles of the multiple copies of genes involved in the oxidation of NH3 in N. europaea. Mutagenesis made possible the study of NirK in N. europaea and helped us to understand its roles in NO and N2O generation (Beaumont et al., 2002; Cantera and Stein, 2007). Genes in the metabolism of carbon were also studied by mutagenesis (Hommes et al., 2006). In the metabolism of NH3, N. europaea utilizes an array of Fe- and heme-containing proteins (Upadhyay et al., 2003; Whittaker et al., 2000). Mutagenesis made possible the study of the Fe uptake regulator, Fur (Arp laboratory, unpublished), and to characterize the specificity of the only annotated ABC-type siderophore transporter (Vajrala et al., 2010) and the ferrioxamine siderophore transporter (Wei et al., 2007).

The protocols described here should be equally applicable to other AOB isolates, although the length of mutant recovery times may be considerably longer for slower growing strains like Nitrosomonas oligotropha. One should keep in mind that growing conditions for other AOB isolates are frequently different from N. europaea, so substrate concentration, temperature, pH, and other limitations must be considered. Ultimately, to be useful, the inactivation of a gene ideally should produce a phenotype that is neither lethal nor hampers growth considerably.

Further development of AOB transformation methodology includes high-throughput screening approaches for selectable phenotypes, which would allow for random mutagenesis and pathway discovery. As described above, the largest hurdles to overcome include the slow growth rates and limited substrate use of AOB that precludes use of most techniques developed for heterotrophic bacteria. Recent developments in robotic liquid handling systems and single cell technologies may help overcome these limitations.

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Genetic Determination of Octopine Degradation

P.M. KLAPWIJK, R.A. SCHILPEROORT, in Molecular Biology of Plant Tumors, 1982

II OCTOPINE AND TUMOR FORMATION

The group of Morel in France found that there exists a remarkable relation between the tumor inducing bacterium and the opine content of the resulting crown gall: strains inducing the synthesis of octopine are able to degrade octopine and to utilize it as a nitrogen source, whereas nopaline inducers are able to degrade and to utilize nopaline (Petit et al., 1970; Lippincott et al., 1973). Two exceptional strains were thought to degrade both octopine and nopaline, but later it became clear that this had been due to contamination (Petit and Tempé, 1978). The positive correlation between the degradative capacity of a strain and the strain-specific opine synthesis readily led Morel and his associates to the idea that gene transfer might occur during the induction of a tumor. Provided that, as in the mollusk Pecten maximus (Van Thoai and Robin, 1961) the synthesis and breakdown of octopine could be carried out by a single enzyme, it was conceivable that the bacterial genes coding for this enzyme were transferred into the plant cell. In the plant, the reaction would be reverted to synthesis as a function of the local chemical environment. This constituted a simple hypothesis, which could be tested by genetic experiments (Petit et al., 1970; Morel, 1971).

A way to perform this testing was to isolate bacterial mutants unable to degrade octopine.* If such mutants would induce tumors without octopine synthesis, this would indicate that the genes responsible for the degradation of octopine in the bacterium are the same as the genes controlling octopine synthesis in the tumor. Mutants unable to utilize octopine have been obtained in different ways. First, in a direct way by the isolation of mutant clones unable to grow substantially with octopine as a sole source of nitrogen or as a source of arginine (Klapwijk et al., 1976; Montoya et al., 1977). Second, by selection of homooctopine resistant clones (Petit and Tempé, 1978; Klapwijk et al., 1978). Homooctopine [N2-(d-1-carboxyethyl)-l-homoarginine; Fig. 1] is as a structural analog of octopine, which is toxic for cultures of A. tumefaciens with an induced or a constitutive level of octopine degrading enzymes. This toxic effect presumably results from the production of homoarginine. Therefore, when a population of bacteria is exposed to homooctopine, only those cells that are missing one or both of the enzymes that are involved in octopine degradation—permease and oxidase—will remain unaffected and survive. Dozens of mutants have been recovered from these various procedures. They shared one property: when they induced crown galls, these tumors contained octopine (Klapwijk et al., 1976, 1978; Montoya et al., 1977; Petit et al., 1978b). Although not refuting the gene transfer hypothesis, this finding obviously provides evidence that the genes controlling octopine synthesis in the plant tumor and the bacterial octopine degradation genes are different and do not code for the same enzyme. This distinction is in accordance with biochemical data. Octopine degradation is carried out by a membrane-bound and cytochrome-linked oxidase, whereas its synthesis is carried out by a cytosol enzyme dependent on NADPH (Jubier, 1975; Bomhoff, 1974; Lejeune, 1973; Hack and Kemp, 1977; Otten et al., 1977).

As will be dealt with more extensively below, the genes controlling octopine and nopaline metabolism are located on the Ti plasmids which are present in all virulent A. tumefaciens strains. The mapping data presently available for octopine and nopaline Ti plasmids are also indicative of a physical separation of the genes controlling degradation and the genes controlling synthesis (Koekman et al., 1979; Schell, 1978). Finally, two exceptional mutant strains have been isolated that induced tumors without octopine (Klapwijk et al., 1978). This was not at variance with the results discussed above, because it could be demonstrated that these strains carried Ti plasmids that had suffered large deletions resulting in the loss of synthesis as well as degradation genes (Koekman et al., 1979).

The genetic and biochemical evidence obtained in several laboratories led to three conclusions: (1) The gene transfer hypothesis for crown gall tumorigenesis cannot be confirmed directly by the genetic approach using the octopine connection. (2) The bacterial capacity to degrade opines does not play an essential role in crown gall tumor formation. (3) The synthesis of opines in the tumor tissue is not a condition for tumor growth (see also the other chapters of this treatise).

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INTERCONVERSION OF PURINE RIBONUCLEOTIDES

J. Frank Henderson, A.R.P. Paterson, in Nucleotide Metabolism, 1973

C NUCLEOTIDE INTERCONVERSION IN BACTERIAL MUTANTS

The above-mentioned reactions of adenylate and guanylate synthesis have been confirmed by the use of bacteria and bacterial mutants which require specific purines for growth.

Some mutants require either xanthine or guanine for growth and have been shown to lack the enzyme responsible for the conversion of inosinate to xanthylate.

Other mutants require guanine specifically and excrete xanthosine; they lack the second enzyme in this pathway.

In neither case can the mutants grow on adenine or hypoxanthine, thereby demonstrating the essential character of this pathway of guanylate synthesis.

Among mutants which require adenine for growth, some have been shown to lack the enzyme which makes adenylosuccinate, and others, that which cleaves it to adenylate. The fact that the loss of either of these enzymes results in a specific requirement for adenine proves the essential nature of these steps for the production of adenylate from ribonucleotides of hypoxanthine, xanthine, and guanine.

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Culture Collections

K.E. Sanderson, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Ownership of Cultures and Use of Material Transfer Agreements

Biological materials including mutant strains may have considerable commercial value, and many biological materials, including bacterial strains and mutants, have been patented. This can have the effect of limiting the ability to exchange biological materials. Many institutions require material transfer agreements (MTAs) before agreeing to provide biological materials to requesting institutions. In general, the genetic stock centers discussed here take into their collections only materials that are made available to any requestor; the original depositor gives up all rights of ownership, and no MTA is required of the requestor. The genetic stock centers usually require payment of a fee and request that publication resulting from use of the culture(s) should acknowledge the culture collection.

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Bacteriophages, Part A

Graham F. Hatfull, in Advances in Virus Research, 2012

6 Mycobacterial recombineering

Recombineering [genetic engineering using recombination (Court et al., 2002)] offers a general approach to constructing mutant bacterial derivatives by taking advantage of the high frequencies of homologous recombination that can be accomplished by the expression of phage-encoded recombination systems. Perhaps the most widely used system in E. coli is the λ-encoded Red system in which three proteins, Exo, Beta, and Gam, contribute to recombination proficiency. Exo is an exonuclease that degrades one strand of dsDNA substrates, Beta is a protein that promotes pairing of complementary DNA strands, and Gam is an inhibitor of RecBCD (Court et al., 2002). When either dsDNA or short ssDNA substrates are introduced into E. coli by electroporation, recombination with a chromosomal or plasmid target occurs efficiently; in some configurations, desired recombinants can be identified even without selection. Similar systems have been described that utilize the RecET system encoded by the E. coli rac prophage (Murphy, 1998; Zhang et al., 1998).

The E. coli recombineering systems do not function well in mycobacteria, especially when using dsDNA substrates (van Kessel and Hatfull, 2007, 2008a). Mycobacterial-specific recombineering systems have been developed using mycobacteriophage-encoded recombinases, especially those related to the RecET systems (van Kessel and Hatfull, 2007, 2008a,b; van Kessel et al., 2008), such as genes 60 and 61 of phage Che9c (Fig. 12). When both Che9c gp60 and gp61 are expressed from an inducible expression system in M. smegmatis or M. tuberculosis, recombination frequencies are elevated substantially. Introduction of a dsDNA allelic exchange substrate in which 500–1000 bp of chromosomal homology flank an antibiotic resistance marker, followed by selection, generates recombinants efficiently (van Kessel and Hatfull, 2007). dsDNA recombineering works well and reproducibly in M. smegmatis, but anecdotal reports suggest that it may be somewhat more erratic in M. tuberculosis, perhaps due to irreproducibility of efficient expression of the recombinases.

Recombineering using ssDNA substrate requires only short synthetic oligonucleotide-derived substrates, provided that mutations are introduced that confer a selectable phenotype (van Kessel and Hatfull, 2008a). Interestingly, in both M. smegmatis and M. tuberculosis there is a very substantial strand bias, such that oligonucleotides with complementary sequences can yield recombinants at frequencies differing by more than 104-fold (van Kessel and Hatfull, 2008a). For engineering purposes it is therefore important that the most efficient of the two possible oligonucleotides is used, which is usually that corresponding to the leading strand of chromosomal DNA replication (i.e., can base pair with the template for lagging strand synthesis). ssDNA recombineering can be used to generate recombinants in the absence of direct selection using coelectroporation of two oligonucleotides: one designed to introduce the desired mutation and one that can be used for selection. A high proportion of selected recombinants also carry the unselected mutation and can be detected by physical screening (van Kessel and Hatfull, 2008a).

Recombineering provides an especially powerful tool for genetic manipulation of the mycobacteriophages themselves (Marinelli et al., 2008; van Kessel et al., 2008). The Bacteriophage Recombineering of Electroporated DNA (BRED) system involves coelectroporation of a phage genomic DNA substrate and a short (∼ 200 bp) dsDNA substrate in a strain in which recombineering functions have been induced. Plaques can then be recovered on solid media in an infectious center configuration in which each electroporated cell that has taken up phage DNA gives rise to a plaque. When individual plaques are screened for the presence of either wild-type or mutant alleles at the targeted site, all contain the wild-type allele, but 10% or more also contain the mutant allele (Marinelli et al., 2008). The desired phage mutant can then be recovered from this mixed primary plaque by replating and testing individual secondary plaques. In this way, two rounds of polymerase chain reaction analysis of 12–18 plaques typically generates the desired mutant, provided that the mutant is viable. In at least some cases, nonviable plaques can be recovered by complementation (Marinelli et al., 2008; Payne et al., 2009). BRED can be used to introduce insertions, deletions, and point mutations into mycobacteriophage genomes (Marinelli et al., 2008).

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Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Chemical, Optical and Bioorthogonal Methods

Kirsten Deprey, Joshua A. Kritzer, in Methods in Enzymology, 2020

2.1 Overview of CAPA

CAPA takes advantage of the HaloTag protein, originally developed by Wood and co-workers (Los et al., 2008). HaloTag is a mutant bacterial haloalkane dehalogenase that has been modified to react irreversibly with a chloroalkane ligand. The chloroalkane fits into the deep and narrow active site of HaloTag, while anything attached to the chloroalkane tag remains outside the pocket (Fig. 1A). The reaction between HaloTag and the chloroalkane is bioorthogonal and proceeds with rapid kinetics (Encell et al., 2012; Los et al., 2008). CAPA uses a cell line that stably expresses HaloTag exclusively in the cytosol, anchored to the cytosolic face of the outer mitochondrial membrane (Ballister, Aonbangkhen, Mayo, Lampson, & Chenoweth, 2014).

Fig. 1. The chloroalkane penetration assay (CAPA). (A) Structure of ligand-bound HaloTag (PDB 5UXZ), with only the linker shown (Liu et al., 2017). Overlay shows a commonly used linker and its relative positioning within the HaloTag active site; blue sphere denotes a molecule-of-interest attached to the chloroalkane and linker. HaloTag is expressed in the cytosol of the CAPA cell line and reacts covalently with chloroalkane-tagged molecules in a deep active site pocket. (B) Schematic of CAPA. Cells are pulsed with a ct-molecule, chased with ct-dye, and analyzed by flow cytometry. (C) The mean red fluorescence of the histogram of 5000 cells at each concentration is plotted as a single point on a plot of red fluorescence versus concentration of ct-molecule. The dose dependence typically follows a sigmoidal curve, and an IC50 curve fit is used to calculate the midpoint (CP50) value.

In CAPA, the HaloTag-expressing cell line is pulsed with a chloroalkane-tagged molecule of interest ("ct-molecule"). If the ct-molecule reaches the cytosol, it will react with HaloTag and block a portion of HaloTag active sites. A subsequent chase with a chloroalkane-tagged dye ("ct-dye") allows the ct-dye to react with all remaining open HaloTag active sites (Fig. 1B). The cells are then washed to remove unreacted dye, trypsinized, and analyzed by flow cytometry to measure the red fluorescence from the ct-dye (Peraro et al., 2018). The more cell-penetrant the molecule, the less red fluorescence that will be detected. Because the assay can be performed in a 96-well plate using small volumes, we typically test serial dilutions of chloroalkane-tagged molecules in parallel to provide a dose-dependence curve of cytosolic localization (Fig. 1C). In this way, the assay offers relative but precise quantitation of the extent of cytosolic localization of any ct-molecule of interest.

CAPA reads out HaloTag occupancy as a proportional, but indirect, measure of cytosolic localization. In an early control experiment, we sought to validate that decreased CAPA signal indeed correlates to a proportional increase in ct-molecule that covalently reacted with cytosolic HaloTag (Peraro et al., 2018). In this experiment, we performed CAPA with a ct-biotin, and we also lysed a portion of the cells after incubation with ct-biotin to analyze the HaloTag-biotin covalent adduct. After lysing the cells, we pulled down the HaloTag-biotin adduct with streptavidin beads and performed a Western blot to detect the HaloTag construct. We directly compared CAPA signal to degree of HaloTag biotinylation, and verified that CAPA signal was indeed inversely proportional to the degree to which HaloTag was modified by the exogenously added ct-molecule (Peraro et al., 2018).

To simplify discussion, in subsequent sections we will use "ct-molecule" or "ct-functional group" to denote a chloroalkane-tagged molecule or functional group, such as "ct-Tat" or "ct-COOH."

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PYRIMIDINE RIBONUCLEOTIDE SYNTHESIS FROM BASES AND RIBONUCLEOSIDES

J. Frank Henderson, A.R.P. Paterson, in Nucleotide Metabolism, 1973

Publisher Summary

This chapter presents pyrimidine ribonucleotide synthesis from bases and ribonucleosides. It was not evident whether preexisting pyrimidines or their derivatives could be utilized for nucleic acid synthesis. The discovery of bacterial mutants with absolute requirements for pyrimidines made it apparent that alternatives to the de novo synthetic route existed, at least in microorganisms. Pyrimidine utilization was more difficult to demonstrate in animals. Because of the capability to synthesize pyrimidines de novo, the traditional type of nutrition experiment could not be employed and the matter remained unresolved until isotopically labeled pyrimidines became available. The chapter also discusses metabolism of pyrimidine bases and their ribonucleosides and utilization of pyrimidine nucleosides. It explains the pathways by which pyrimidine bases and ribonucleosides are metabolized.

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