Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression.

vir regulon expression in Agrobacterium tumefaciens involves both chromosome- and Ti-plasmid-encoded gene products. We have isolated and characterized a new chromosomal gene that when mutated results in a 2- to 10-fold reduction in the induced expression of vir genes by acetosyringone. This reduced expression occurs in AB minimal medium (pH 5.5) containing either sucrose or glucose and containing phosphate at high or low concentrations. The locus was cloned and used to complement A. tumefaciens strains harboring Tn5 insertions in the gene. Sequence analysis of this locus revealed an open reading frame with strong homology to the miaA locus of Escherichia coli and the mod5 locus of Saccharomyces cerevisiae. These genes encode tRNA: isopentenyltransferase enzymes responsible for the specific modification of the A-37 residue in UNN codon tRNA species. The function of the homologous gene in A. tumefaciens was proven by genetic complementation of E. coli miaA mutant strains. tRNA undermodification in A. tumefaciens miaA mutant strains may reduce vir gene expression by causing a reduced translation efficiency. A slight reduction in the virulence of these mutant Agrobacterium strains on red potato plants, but not on tobacco, tomato, kalanchoe, or sunflower plants, was observed.     

Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting

Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.     

Improvement of reading frame maintenance is a common function for several tRNA modifications

Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild-type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A-site and thereby increasing peptidyl-tRNA slippage; or (ii) a hypomodified peptidyl-tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.     

tRNA modification activity is necessary for Tet(M)-mediated tetracycline resistance.

Tet(M) protein interacts with the protein biosynthetic machinery to render this process resistant to the tetracycline in vivo and in vitro (V. Burdett, J. Biol. Chem. 266:2872-2877, 1991). To understand this process more completely, a mutant of Escherichia coli which is altered in the ability of Tet(M) to confer resistance has been identified. This mutation maps to miaA and displays phenotypes characteristic of previously isolated miaA mutations. The miaA gene product modifies A37 adjacent to the anticodon of several tRNA species. Both the mutant isolated in this work and previously isolated miaA mutants confer tetracycline sensitivity in the presence of functional Tet(M), both share a slow growth phenotype, and in neither case is a wild-type phenotype restored in trans by F'112 carrying the 89- to 98-min region of the chromosome. These similar phenotypes further substantiate the assignment of the mutation described here to the miaA locus.     

Reexamining the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium tumefaciens strain C58

Isogenic strains of Agrobacterium tumefaciens carrying pTiC58, pAtC58, or both were constructed and assayed semiquantitatively and quantitatively for virulence and vir gene expression to study the effect of the large 542-kb accessory plasmid, pAtC58, on virulence. Earlier studies indicate that the att (attachment) genes of A. tumefaciens are crucial in the ability of this soil phytopathogen to infect susceptible host plants. Mutations in many att genes, notably attR and attD, rendered the strain avirulent. These genes are located on pAtC58. Previous work also has shown that derivatives of the wild-type strain C58 cured of pAtC58 are virulent as determined by qualitative virulence assays and, hence, pAtC58 was described as nonessential for virulence. We show here that the absence of pAtC58 in pTiC58-containing strains results in reduced virulence but that disruption of the attR gene does not result in avirulence or a reduction in virulence. Our studies indicate that pAtC58 has a positive effect on vir gene induction as revealed by immunoblot analysis of Vir proteins and expression of a PvirB::lacZ fusion.     

Identification and characterization of ispA, a Shigella flexneri chromosomal gene essential for normal in vivo cell division and intercellular spreading

The virulent phenotype of Shigella requires loci on the chromosome as well as on the large virulence plasmid, and is regulated via a complex web of interactions amongst various chromosomal and large plasmid genes. To further investigate the role of chromosomal loci in virulence, we performed random Tn 10 mutagenesis in Shigella flexneri YSH6000T, and isolated an avirulent mutant (V3404) incapable of spreading throughout an epithelial cell monolayer. Although V3404 initially spread intercellularly at the same rate as the wild-type, it gradually slowed down and ceased spreading as a result of increasing defects in cell division, leading to the formation of long filamentous bacteria lacking septa, trapped within cells. In addition, the mutation affected the ability of V3404 to polymerize actin, a prerequisite for intra- and inter-cellular spreading ability. Sequencing of Tn 10 -flanking DNA revealed that the mutated gene, designated ispA (intracellular septation), was equivalent to a previously sequenced but uncharacterised gene of Escherichia coli located between trp and tonB . Using E. coli sequence data, we cloned the ispA gene from the YSH6000T chromosome and found that it complemented the V3404 mutation. Nucleotide sequencing and in vitro expression experiments revealed that ispA coded for a small (21 kDa), very hydrophobic protein. These results thus show that ispA is an essential virulence gene affecting several functions of the virulence process.     

Reaction of the hematopoietic system in mice to the administration of virulent and avirulent strains of Shigella flexneri 2A

The influence of live bacteria and antigenic preparations of virulent and avirulent stains of Shigella flexneri 2a on the endogenous splenic colony formation in murine hematopoietic cells has been studied. The different stimulating activity of live microbial cells of virulent and avirulent strains Shigella flexneri 2a and their antigenic preparations on the endogenous colony formation has been shown. The effect observed depends on the preparation doses and time of their administration before irradiation of the animals. The stimulating influence on the development of hemopoiesis endogenous foci may be conditioned by the action of heat-labile products of the live microbial cells and closely correlates with the virulence of strains studied.