Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium

Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.     

Evolution of Transposons: Natural Selection for Tn5 in ESCHERICHIA COLI K12

A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.     

Global analysis of the genes involved in the thermotolerance mechanism of thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Transposon insertion in the ftsK gene impairs in planta growth and lesion-forming abilities in Pseudomonas syringae pv. syringae B728a

A Tn5 insertion in the ftsK gene of Pseudomonas syringae pv. syringae B728a impaired brown spot lesion formation on Phaseolus vulgaris, the ability to grow within bean leaves, and swarming ability on semisolid agar. Plasmids containing the ftsK gene were sufficient to complement the original Tn5 mutant for lesion formation and swarming and partially restored in planta growth.     

Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA.

We report the unexpected result that Escherichia coli isolates containing a multicopy plasmid (pDT1.5) carrying the manganese-superoxide dismutase gene sodA were more sensitive than the wild type to paraquat-mediated growth inhibition. The pDT1.5 locus responsible for the paraquat-sensitive phenotype was delimited to a 0.6-kilobase segment by transposon Tn5 mutagenesis. Moreover, superoxide dismutase activity was the same as in the wild type in strains carrying pDT1.5::Tn5 insertions mapping to the 0.6-kilobase locus. These data identify the 0.6-kilobase segment as the locus of sodA and establish an association between growth inhibition by paraquat and the function of the plasmid-borne sodA gene.     

Structural and physiological characteristics of some sheathed bacteria

The fine structure and growth of two gram-negative and two gram-positive strains of sheathed bacteria, isolated from activated sludge, have been studied. Their anatomy is quite different fromSphaerotilus natans. The cells are rectangular, tightly joined together, non-motile and enclosed by a sheath which is thin in the gram-negative strains and rather electron-dense in the two larger gram-positive strains. The four strains grow slowly and form pink filamentous flocs in liquid media.     

Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.     

Isolation and characteristics of compound transposons Tn1000::Tn5

Two types of compound transposons were derived. In the first case, transposon Tn5 is inserted into the gene responsible for Tn1000 transposase synthesis. In the other, Tn5 is inserted into the region near the left end of Tn1000, where no functionally significant genes were found. It is known that translocation of the compound transposons does not depend on their size and takes place with the efficiency close to that characteristic of the intact Tn1000. Insertion of Tn5 into the gene coding for Tn1000 transposase results in sharp decrease in the efficiency of Tn1000 translocations. This effect, however, may be eliminated by introduction into the cell of the intact Tn1000.     

Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant

We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition. Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media. Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition. In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements. Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain. Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions. We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R. The mutation altered the amino acid sequence of both transposase and inhibitor.     

dsg, a gene required for Myxococcus development, is necessary for cell viability.

Previous work identified the dsg gene as necessary for cell-cell interaction in Myxococcus xanthus. Point mutations of this gene, such as dsg-439, are viable, but insertions of Tn5 within the dsg gene (dsg::Tn5) are lethal. Partial diploids, dsg::Tn5/dsg+ or dsg::Tn5/dsg-429 or dsg::Tn5/dsg-439, are also viable, showing that the lethal effect of the haploid insertions is due to loss of function. Thus the evidence implies that the dsg gene is essential for viability as well as development, but its essential quality differs between growth and development because dsg-429 and dsg-439 mutants grow normally, but are unable to develop.     

Megaplasmid pRme2011a of Sinorhizobium meliloti is not required for viability

We report the curing of the 1,360-kb megaplasmid pRme2011a from Sinorhizobium meliloti strain Rm2011. With a positive selection strategy that utilized Tn5B12-S containing the sacB gene, we were able to cure this replicon by successive rounds of selecting for deletion formation in vivo. Subsequent Southern blot, Eckhardt gel, and pulsed-field gel electrophoresis analyses were consistent with the hypothesis that the resultant strain was indeed missing pRme2011a. The cured derivative grew as well as the wild-type strain in both complex and defined media but was unable to use a number of substrates as a sole source of carbon on defined media.     

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.     

Overinitiation of chromosome replication in the Escherichia coli dnaAcos mutant depends on activation of oriC function by the dam gene product

The activity of DnaA protein, the initiator of chromosome replication in Escherichia coli , is regulated by adenine nucleotide binding; the ATP-bound form, not the ADP-bound form, is active. DnaAcos is a mutant protein that is insensitive to negative regulation by ADP. Initiation of chromosome replication occurs excessively in the dnaAcos mutant at 30°C, a restrictive temperature for growth. To determine the control factors that act independently of adenine nucleotide binding of DnaA, we analysed suppressors from the dnaAcos mutant isolated by Tn 5 insertion mutagenesis. Three of the suppressors carried Tn 5 in the aroK or aroB gene, the first two cistrons in the dam operon. Complementation tests revealed that the dam gene is responsible for the suppression. Over-replication of the chromosome was inhibited in the dnaAcos aroK ::Tn 5 double mutant, and initiation of chromosome replication in the dnaA ⁺ aroK ::Tn 5 mutant was partially inhibited. The aroK  (or B  )::Tn 5 cells contained DnaA molecules at a level similar to that in the parental aroBK  ⁺ strain. Moreover, dnaAcos suppression depended on the function of the seqA gene. Thus, Dam activity positively regulates initiation of chromosome replication in vivo . SeqA function seems to be distinguished from the control of DnaA protein by adenine nucleotide binding.     

Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus

In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used Tn5::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposon-harboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutorphus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of Tn5 were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a lambda L47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of Tn5 covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of Tn5::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.     

A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin.

A plasmid shuttle vector for Escherichia coli and mycobacteria was constructed from an E. coli plasmid containing the ColE1 origin, a 2.6-kb PstI fragment from bacteriophage D29 that grows in numerous mycobacterial species, and the kanamycin resistance gene either of Tn903 or of Tn5. The resultant plasmid is 7.63 kb and can be introduced via transformation into Mycobacterium smegmatis with high efficiency. In M. smegmatis the plasmid is stable and apparently present in multiple copies. Bioluminescence (luxA and luxB of Vibrio harveyi and fischeri) has been expressed in M. smegmatis from the aminoglycoside transferase promoter of Tn5. The D29 fragment should carry an origin of replication and some associated genes that act on it since various mutations destroy the ability of this fragment to replicate in M. smegmatis. The fragment was localized on the D29 genome map.     

以转座子Tn5作弗氏中华根瘤菌的可识别生态学标记的研究──Tn5的水平转移及其对R．fredii Tn5突变株运动的影响

将一株弗氏中华根瘤菌（Ｒ．ｆｒｅｄｉｉ）ＱＢ１１３０的Ｔｎ５插入突变株ＯＮ－２用于生态学研究,以评估Ｔｎ５在自然环境中的水平转移以及各种水势下Ｔｎ５对突变株ＯＮ－２在土壤中运动的影响．试验表明,在自然潮湿的土壤中,Ｔｎ５本身的水平转移频率很低,且与Ｔｎ５插入相关的突变株卡那霉素抗性表型标记在非选择性平板上连续传４０代后仍然稳定．突变株ＯＮ－２与相对应的野生型菌株ＱＢ１１３０在各种相同水势的土壤中的运动无明显差异（Ｐ＝０．０１）,表明Ｔｎ５的插入不影响突变株的运动．因此,Ｔｎ５可作为研究Ｒ．ｆｒｅｄｉｉ基因工程菌大回应用的一个稳定有效的生态学标记．