Genetic Analysis of Flagellar Mutants in Escherichia coli

Flagellar mutants in Escherichia coli were obtained by selection for resistance to the flagellotropic phage chi. F elements covering various regions of the E. coli genome were then constructed, and, on the basis of the ability of these elements to restore flagellar function, the mutations were assigned to three regions of the E. coli chromosome. Region I is between trp and gal; region II is between uvrC and aroD; and region III is between his and uvrC. F elements carrying flagellar mutations were constructed. Stable merodiploid strains with a flagellar defect on the exogenote and another on the endogenote were then prepared. These merodiploids yielded information on the complementation behavior of mutations in a given region. Region III was shown to include at least six cistrons, A, B, C, D, E, and F. Region II was shown to include at least four cistrons, G, H, I, and J. Examination of the phenotypes of the mutants revealed that those with lesions in cistron E of region III produce "polyhooks" and lesions in cistron F of region III result in loss of ability to produce flagellin. Mutants with lesions in cistron J of region II were entirely paralyzed (mot) mutants. Genetic analysis of flagellar mutations in region III suggested that the mutations located in cistrons A, B, C, and E are closely linked and mutations in cistrons D and F are closely linked.     

Transductional analysis of the flagellar genes in Pseudomonas aeruginosa.

Complementation in bacteriophage E79 tv-l-mediated transduction and the phenotypic properties of the flagellar genes in Pseudomonas aeruginosa PAO were investigated by using 195 flagellar mutants of this organism. A total of 15 fla. 1 mot, and 2 che cistrons were identified. At least 5 fla cistrons (fla V to flaZ) and one mot cistron resided in one region, and at least 10 fla cistrons (flaA to flaJ) and two che cistrons (cheA and cheB) resided in another. The flaC mutants exhibited cistron-specific leakiness on motility agar plates. The flaE cistron may be the structural gene for the component protein of the flagellar filament. The cheA mutations, which resulted in pleiotropic phenotypes for flagellar formation, motility, and taxis, belonged to the same complementation group as the flaF mutations; that is, we inferred that cheA and flaF are synonymous.     

Identification of cistrons involved in conjugal transfer of narrow-host-range R plasmid R91-5 of Pseudomonas aeruginosa.

The development of a transductional method for complementation tests between transfer-deficient mutants of the narrow-host-range R plasmic R91-5 of Pseudomonas aeruginosa has allowed the indentification of cistrons involved in the conjugal transfer of this plasmid. Complementation tests performed between transfer-deficient mutants characterized phenotypically with respect to sensitivity to donor-specific phage, ability to inhibit the replication of phage G101, and expression of entry-exclusion has identified a minimum of 10 transfer cistrons. Although most mutagen-induced mutants were relatively heterogeneous and appeared to be affected in a single cistron only, a high proportion of mutants isolated after selection for donor-specific phage resistance had deletions but always included tra Y. Mutants selected directly on the basis of transfer deficiency which also became donor-specific phage resistant fell into all 10 cistrons, suggesting that many R91-5 transfer cistrons are concerned with the synthesis of sex pili and other surface structures necessary for conjugal transfer. Conversely, most retaining donor-specific phage sensitivity belonged to one cistron, whereas transfer-deficient mutants which had also lost the ability to inhibit the replication of phage G101 comprised four cistrons.     

Slow motile mutant in Salmonella typhimurium

Enomoto, Masatoshi (National Institute of Genetics, Misima, Japan). Slow motile mutant in Salmonella typhimurium. J. Bacteriol. 90:1696-1702. 1965.-A slow motile mutant, SJ399, was isolated from a wild-type strain of Salmonella typhimurium TM2. The mutant was as motile as the wild type in broth culture at 37 C. However, on semisolid medium it produced a much narrower swarming band than TM2. The motility of this mutant was hindered by the viscosity of semisolid medium. H antigenicity and morphological characters of flagella of the mutant were the same as those of the wild type. The motility phage, chi, responded differently to SJ399 and the wild type. Plaques of SJ399 were small and cloudy, whereas on the wild type they were large and clear. The efficiency of plating on SJ399 was 0.36 as compared with 1 with the wild type. Stained preparations revealed that the mutant had about one-third the number of flagella of the wild type. The reduction of the number of flagella also was ascertained by biochemical measurement of flagellar protein which was purified after deflagellation from cells. The content of flagellin in SJ399 was about 32% of that of the wild type. Phage P22-mediated transductions from SJ399 to nonflagellated (fla(-)) and paralyzed (mot(-)) mutants showed that the mutant SJ399 complements seven fla(-) and three mot(-) strains which are representative mutants of flagellation and motility cistrons, respectively. The mutation site of SJ399 was cotransduced with both motA and B cistrons. The two point cross tests between SJ399 and mot mutants revealed that the mutation site of SJ399 is located in the motB cistron. The insertion of the genetic region containing the mutation site of SJ399 to the motB cistron is discussed in relation to intracistronic complementation.     

Gene Expression During the Development of Bacteriophage φ29 III. Analysis of Viral-Specific Protein Synthesis with Suppressible Mutants

Fifty-four suppressible mutants of bacteriophage phi29 have been isolated with a variety of mutagens and assigned to eight complementation groups. Viral-specific protein synthesis in UV light-irradiated, nonsuppressing Bacillus subtilis 60084 was analyzed with exponential acrylamide gels. Four additional phi29 proteins which were undetected on ordinary acrylamide gels are reported in this paper. Five phage phi29 proteins have been unambiguously assigned to specific cistrons. Two cistrons had pleiotropic effects on viral protein synthesis. Mutants in cistrons I or II were unable to synthesize DNA in nonsuppressing bacteria. Mutants in cistron I were unable to attach viral chromosomes to the host cell membrane, and the protein responsible for this function has been identified. The other viral protein playing a role in phage phi29 DNA synthesis is also identified and assigned to cistron II. Mutants in cistron II can attach viral chromosomes to membrane, but cannot synthesize DNA in nonsuppressing bacteria.     

Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1

Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region. The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping. The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb. Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five. The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion. Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region.     

Cotransduction of strA and Ribosomal Protein Cistrons in Escherichia coli-Salmonella typhimurium Hybrids

Genetic mapping of ribosomal protein cistrons of Salmonella typhimurium and Escherichia coli was performed by phage P1 mediated, generalized transduction. From an E. coli hybrid strain which carried a S. typhimuirum F' factor, an E. coli strain was constructed which had integrated S. typhimurium genetic material including the region of the strA locus. Salmonella genetic material from this hybrid was transduced into E. coli recipients. The ribosomal protein electrophoretic patterns of these hybrid transductants were correlated with the presence of markers contributed by each parent.The results of these studies indicate that cistrons for at least three characteristic S. typhimurium and two E. coli 30S ribosomal proteins are closely linked to the strA locus on the genetic maps of both organisms. At least one cistron coding for a 50S ribosomal protein is also closely linked to this locus on both maps. These findings support the concept that cistrons coding for the ribosomal proteins are clustered in one area of the genome. Mutations to spectinomycin and streptomycin resistance are closely linked in S. typhimurium and are located at strA.     

Genes for the hook-basal body proteins of the flagellar apparatus in Escherichia coli.

Of the more than 30 genes required for flagellar function, 6 are located between pyrC and ptsG on the Escherichia coli genetic man. This cluster of genes is called flagellar region I. Four-point transductional crosses were used to establish the position and order of the region I flagellar genes with respect to the outside markers ptsG and pyrC. Bacteriophage lambda-E. coli hybrids that contained most of the genes necessary for flagellar formation were constructed. The properties of specific hybrids that carried the region I fla genes were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the lambda hybrids demonstrated the existence of at least six flagellar-specific cistrons. These directed the synthesis of polypeptides with the following apparent molecular weights: flaV, 11,000; flaK, 42,000; flaL, 30,000 and 27,000; flaM, 38,000; flS, 60,000; and flaT, 35,000. Plasmid ColE1-E. coli hybrids with region I flagellar genes were also used to program the synthesis of polypeptides in minicell-producing strains. The polypeptides synthesized in these experiments were identical to polypeptides of the hook-basal body structure and helped to confirm the assignment of genes to specific polypeptides. The synthesis of all of these polypeptides was regulated by the same mechanism that regulates the synthesis of other flagellar-related structural components.     

Process of Infection with Bacteriophage φX174: XXXIV. Kinetics of the Attachment and Eclipse Steps of the Infection

The products of phiX cistrons II, III, and VII are demonstrated to affect the attachment of the phage to its host Escherichia coli C; therefore, by inference, these cistrons influence, directly or indirectly, the structure of proteins in the virus particle. Two of the mutations which alter attachment kinetics, ts79 in cistron III and h in cistron VII, also affect the electrophoretic mobility of the virus and emphasize the role of charge in the attachment interaction with the host. The kinetics for attached phage to go into "eclipse" are first-order and biphasic; about 85% of the phage eclipse at one rate (k(e) = 0.86 min(-1)) and the remainder do so at a distinctly lower rate (k(e) = 0.21 min(-1)). No phiX cistrons yet identified affect the eclipse process. The lowest temperature at which eclipse is detected is 19 C. The Arrhenius activation energy for phage eclipse has the high value of 36.6 kcal/mole, indicating the cooperative nature of the event.     

TGATG vector: a new expression system for cloned foreign genes in Escherichia coli cells

A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, employing the effects of translational coupling to optimize expression of cloned cistrons in Escherichia coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon situated on the vector, to form the hybrid operon transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon ('overlappon'). The efficiently translated cro'-cat'-'trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron [the length of the corresponding open reading frame is about 120 amino acids (aa)] includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa) and 3' C-terminal codons of the E. coli trpE gene product. At the 3'-end of the cro'-cat'-'trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C) and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assembly of Bacillus subtilis phage phe29. 2. Mutants in the cistrons coding for the non-structural proteins.

The effect on phage morphogenesis of sus mutations in the cistrons coding for nonstructural proteins has been studied. Mutants in three cistrons analyzed that are involved in phage DNA synthesis, as well as in cistron 16 which codes for a late nonstructural protein, produce prolate capsids which are more rounded at the corners than complete phage heads and have an internal core; they contain the head proteins, the upper collar protein and protein p7, not present in mature phage particles. Mutants in cistron 7 do not produce capsids nor other phage-related structures; this result and the presence of p7 in phage capsids suggest an essential role in capsid assembly for this protein. The protein product of cistron 13 is probably needed for a stable DNA encapsulation since mutants in this cistron produce mainly DNA-free complete phage particles and only about 10% of uninfective DNA-containing complete phage. Cistron 15 codes for a late, partially dispensable, nonstructural protein which is present in the DNA-free capsids produced after infection with the delayed-lysis mutant sus14(1242), used as the wild-type control, or with mutants in cistrons 9, 11,12 and 13. Proteins p15 and p16 are probably involved in the encapsulation of viral DNA in a prohead.     

Construction and characterization of Pseudomonas aeruginosa algB mutants: role of algB in high-level production of alginate.

The algB gene, which is involved in the production of alginate in Pseudomonas aeruginosa, was localized to approximately 2.2 kilobases of DNA from strain FRD by using transposon Tn501 insertion mutagenesis, subcloning, and complementation techniques. The previously reported alg-50(Ts) mutation, which confers the phenotype of temperature-sensitive alginate production, was here designated as an algB allele. A transduction-mediated gene replacement technique was used for site-directed mutagenesis to isolate and characterize algB::Tn501 mutants of P. aeruginosa FRD. Although algB::Tn501 mutants had a nonmucoid phenotype (indicating an alginate deficiency), they still produced about 1 to 5% of wild-type levels of alginate in most growth media and up to 16% in very rich media. The algB::Tn501 mutations had no apparent effect on growth rate or growth requirements. Using another gene replacement technique called excision marker rescue, we constructed a chromosomal algB deletion (delta algB) mutant of P. aeruginosa FRD. The delta algB mutant also produced low levels of alginate as did the algB::Tn501 mutants. The alginate produced by algB::Tn501 mutants resembled wild-type alginate by all criteria studied: molecular weight, acetylation, and proportion of mannuronic and guluronic acids. Thus, the algB gene product is apparently involved in the high-level production of alginate by P. aeruginosa and is not directly involved in the pathway leading to its biosynthesis. Chromosomal mapping of an algB::Tn501 insertion showed linkage to the trp-2 marker on the FRD chromosome as does the algB50(Ts) mutation. The excision marker rescue technique was also used to place the algB::Tn501 marker on the chromosome of characterized strains of P. aeruginosa PAO. The algB::Tn501 mutation mapped near 21 min on the PAO chromosome.     

Two classes of region III flagellar genes in Escherichia coli

We infected various nonflagellated mutants of Escherichia coli with fla-transducing phages and followed the kinetics of the appearance of motility. Our analysis revealed two distinct classes of region III fla genes. Class II fla genes (hag, flaD) functioned 15 min later than class I fla genes (flaN, flaB, flaC, flaO, flaA, flbD, flaQ, flaP) in flagellar morphogenesis. We suggest that the two classes of fla genes are involved in two different stages, initiation (class I) and completion (class II), of flagellar formation.     

Genetic mapping of the G101 bacteriophage (Pseudomonas aeuruginosa by temperature-sensitive mutations

Temperature-sensitive (ts) mutations of the G101 phage were isolated after mutagenesis with hydroxylamine. A complementation analysis of 61ts mutants showed that these mutants may be divided into at least 12 complementation groups. Twots mutants probably originated in genes which control lytic functions of the G101 phage. It was shown by three factor crosses that all of the 12ts mutations tested are localized on that side of the “c” region where the probablecI repressor gene is positioned. Sevents mutations is closely linked to thecI ₂₆ clear marker, three exhibit a closer linkage and two do not exhibit any linkage withcI. All mutations isolated until now can be arrange linearly. According to the present knowledge the preliminary genetic map of the G101 phage is linear.     

Adsorption of Bacillus subtilis bacteriophage PBS 1.

Bacteriophage PBS 1 adsorbs initially on the flagella of its host, Bacillus subtilis (stage I). The phage can adsorb to both active and inactive flagella. Flagellar attachment is nonspecific as PBS 1 was shown to attach to the flagella of Bacillus species other than the normal host B. subtilis. The phage particle then quickly moves down the length of the flagellum to its base, the final adsorption site. Flagellar motion is required for flagellar base attachment (stage II). After proper attachment at the flagellar base, the phage tail sheath contracts sending the tail core through the final adsorption site (stage III). The phage DNA is then injected at this site (stage IV). Stage I adsorption does not cause loss of motility in PBS 1 -- resistant bacilli. The loss of motility observed upon infection of sensitive cells by PBS 1 may be associated with either stage II or stage III of adsorption.     

Genetic and physiological relationships between L-asparaginase I and asparaginase II in Saccharomyces cerevisiae.

The cistron that codes for L-asparaginase I in Saccharomyces cerevisiae (aspl) is not genetically linked to either of the cistrons coding for expression of asparaginase II (asp2 and asp3). Cells containing different combinations of theses enzymes grow at different rates in media in which L-asparagine or D-asparagine is the only source of nitrogen for cell replication. Cells lacking L-asparaginase I but possessing asparaginase II grow more rapidly in medium containing D-asparagine as a nitrogen source than cells containing both enzymes, even though D-asparagine is not a substrate of L-asparaginase I. These results indicate that L-asparaginase I and asparaginase II interact in some way to regulate the utilization of asparagine as a nitrogen source for cell growth.