Identification of the Escherichia coli murI gene, which is required for the biosynthesis of D-glutamic acid, a specific component of bacterial peptidoglycan.

The murI gene of Escherichia coli, whose inactivation results in the inability to form colonies in the absence of D-glutamic acid, was identified in the 90-min region of the chromosome. The complementation of an auxotrophic E. coli B/r strain by various DNA sources allowed us to clone a 2.5-kbp EcoRI chromosomal fragment carrying the murI gene into multicopy plasmids. The murI gene corresponds to a previously sequenced open reading frame, ORF1 (J. Brosius, T. J. Dull, D. D. Sleeter, and H. F. Noller. J. Bacteriol. 148:107-127, 1987), located between the btuB gene, encoding the vitamin B12 outer membrane receptor protein, and the rrnB operon, which contains the genes for 16S, 23S, and 5S rRNAs. The murI gene product is predicted to be a protein of 289 amino acids with a molecular weight of 31,500. Attempts to identify its enzymatic activity were unsuccessful. Cells altered in the murI gene accumulate UDP-N-acetylmuramyl-L-alanine to a high level when depleted of D-glutamic acid. Pools of precursors located downstream in the pathway are consequently depleted, and cell lysis finally occurs when the peptidoglycan content is 25% lower than that of normally growing cells.     

A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules.

We demonstrate for the first time that the broad-host-range stabilization loci from plasmid RK2 cause total retention of plasmids in cells of Rhizobium meliloti during symbiosis with alfalfa. Two derivatives of plasmid RK2, pRK290 and a 7.3-kb mini-RK2 plasmid, were stabilized in R. meliloti cells isolated from root nodules by the insertion of a 3.2-kb DNA fragment or a smaller 0.8-kb DNA fragment derived from the RK2 stabilization region.     

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.     

Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens.

Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine. The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid. The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source. They also utilized mannopinic acid, but not agropine or agropinic acid. In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs. [14C]mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates. These results suggested that the catabolic system of this mutant was not subject to a stringent regulation. A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain. Only the LM136 clone was expressed in the parent Agrobacterium strain A136. Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains. Cells of LM136 utilized [14C]mannopine without generating detectable amounts of intracellular agropine. In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on [14C]mannopine, was in the form of agropine.     

Participation of a cyanobacterial S layer in fine-grain mineral formation.

Cyanobacteria belonging to the Synechococcus group are ubiquitous inhabitants of diverse marine and freshwater environments. Through interactions with the soluble constituents of their aqueous habitats, they inevitably affect the chemistry of the waters they inhabit. Synechococcus strain GL24 was isolated from Fayetteville Green Lake, New York, where it has a demonstrated role in the formation of calcitic minerals. In order to understand the detailed interactions which lead to mineral formation by this organism, we have undertaken detailed ultrastructural studies of its cell surface and the initial events in mineral growth using a variety of electron microscopic and computer image enhancement techniques. Synechococcus strain GL24 has a hexagonally symmetrical S layer as its outermost cell surface component. The constituent protein(s) of this structure appears as a double band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r)s of 104,000 and 109,000. We demonstrate that the S layer acts as a template for fine-grain gypsum and calcite formation by providing discrete, regularly arranged nucleation sites for the critical initial events in the mineralization process. To our knowledge, this is the first time that a bacterial S layer has been shown to have a role in mineral formation in a natural environment, and this report provides conclusive evidence for the specific involvement of bacterial surfaces in natural mineral formation processes.     

A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis.

A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNA(UAGLeu) gene nor its product has been found previously in B. subtilis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtilis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes of B. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.     

Exogenous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants.

The acidic exopolysaccharide (EPS I) produced by Rhizobium meliloti during symbiosis with Medicago sativa has been shown to be required for the proper development of nitrogen-fixing nodules. Cloned DNA from the exo region of R. meliloti is shown to stimulate production of the low-molecular-weight form of this exopolysaccharide, and in this report we show that the symbiotic deficiencies of two exo mutants of R. meliloti, the exoA and exoH mutants, can be rescued by the addition of this low-molecular-weight material at the time of inoculation. For exoA and exoH mutants, rescue with a preparation containing low-molecular-weight exopolysaccharide induces the formation of nitrogen-fixing nodules which appear somewhat later and at a reduced efficiency compared with wild-type-induced nodules; however, microscopic analysis of these nodules reveals similar nodule morphology and the presence of large numbers of bacteroids in each.