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fliL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneousfliL mutant ofS. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the elfect on motility caused by truncation of the gene is probably an indirect one.The flagellar regulons of Escherichia coli and Salmonella typhimurium contain exclusively genes concerned with flagellation, motility, and chemotaxis (10).fliL is the first gene in one of the flagellar operons (3, 7) and is immediately followed by fliM and fliN, two of the three genes responsible for flagellar rotation and switching between the counterclockwise and clockwise directions (13); it was discovered as a result of cloning the adjacent gene, fliM (3, 7), having escaped attention in searches for mutants. FliL is found in the membrane fraction in minicell experiments (3, 11), and on the basis of its sequence, it would be expected to span the membrane just once and have a C-terminal domain in the periplasm. Its role in the flagellar system is unknown.Malakooti et al. (11) found that an E. coli strain, JM7623 (Fig. lb), whose fliL gene had been disrupted by a kanamycin resistance gene (Kmr) cassette, was nonflagellate; they concluded that the mutation was not polar on downstream genes and that the null phenotype for fliL was nonflagellate.We subsequently encountered in S. typhimurium the first example of a spontaneous fliL mutant, SJW2295 (12). This strain carried a severe truncation of the gene yet was motile. Its properties have led us to examine more closely the role of the fliL gene. MATERIALS AND METHODSBacterial strains and plasmids. GM2163 (New England Biolabs, Beverly, Mass.) was used for transformations with DNA containing Clal sites, to avoid dam methylation. UH869 (4) was used as a minicell-producing strain. AW330 (1) and EKK9 (8) are E. coli strains that are wild type for motility and chemotaxis; we used AW330 initially but later changed to EKK9 because we found it had better motility and a higher level of transformation efficiency. E. coli JM7623 (11) (2), with slight modification in the case of the BamHIClaI deletion allele of fliL in plasmid pMR14 (see Results), with which transformation was accomplished by electroporation, and the transformed cells (after 1 h of growth at 30°C to recover) were diluted into 5 ml of Luria broth plus 10 ,ug of chloramphenicol per ml and grown overnight at 44°C to enrich for recombinants before plating. RESULTSProperties of a S. typhimurium mutant with a truncatedfliL gene. Strain SJW2295 (13), a poorly swarming derivat...

In the gap between two closely linked flagellar gene clusters on the Escherichia coi and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coi map), we found an open reading frame whose sequence suggested that it encoded an a-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other ct-amylases were to the excreted liquefying a-amylases of bacilli, with >40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coil gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the ac-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an a-amylase rather than a 1-amylase. The newly discovered gene has been named amyA.The natural physiological role of the AmyA protein is not yet evident.During an investigation of the flagellar genes ofEscherichia coli and Salmonella typhimuium, we encountered a nearby open reading frame whose deduced product sequence suggested that it was that of a cytoplasmic a-amylase.The only major cytoplasmic polysaccharide in these enteric bacteria is glycogen, which is laid down as an energy and carbon reserve, especially under conditions in which carbon is abundant but another major essential element, such as nitrogen, is limiting (27). Under normal growth conditions, glycogen represents only about 2.5% of the dry weight of the cell (26), but it can reach as high as 30% under conditions of carbon abundance and deprivation of another essential element, such as nitrogen (33). A possible role for a cytoplasmic a-amylase might therefore be in glycogen metabolism.The enzymes responsible for glycogen synthesis in E. coli have been studied extensively; they are glucose-i-phosphate adenyltransferase, glycogen synthase, and 1,4-a-glucan branching enzyme. The corresponding structural genes (glgC, glgA, and glgB) are clustered at 76 min on the map (1,2,18).Less is known about the genetics and enzymology of glycogen breakdown in E. coli. By analogy with mammalian systems, one might expect at least an a-glucan phosphorylase and a debranching enzyme. An E. coli protein with a-glucan phosphorylase activity was reported by Chen a...

Targeted mutagenesis and gene knock-out can be mediated by triple helix-forming oligonucleotides (TFO) linked to mutagenic agents, such as psoralen. However, this strategy is limited by the availability of homopurine/homopyrimidine stretches at or near the target site because such sequences are required for high-affinity triplex formation. To overcome this limitation, we have tested TFO conjugated to psoralen via linker arms of lengths varying from 2 to 86 bonds, thereby designed to deliver the psoralen at varying distances from the third strand binding site present at the 3' end of the supFG1 mutation reporter gene. Following triplex formation and UVA irradiation, mutations were detected using an SV40-based shuttle vector assay in human cells. The frequency and distribution of mutations depended on the length of the linker arm. Precise targeting was observed only for linker arms of length 2 and 6, which also yielded the highest mutation frequencies (3 and 14%, respectively). Psoralen-TFO with longer tethers yielded mutations at multiple sites, with the maximum distance from the triplex site limited by the linker length but with the distribution within that range influenced by the propensity for psoralen intercalation at A:T base-pair-rich sites. Thus, gene modification can be extended beyond the site of third strand binding but with a decrease in the precision of the targeting.

Flagellar regions IIIa and IIIb of the Escherichia coli and Salmonella typhimurium chromosomes (at 40 min and 42-43 min, respectively) has been shown to be separated by DNA unrelated to flagellar function, with region IIIa being immediately followed by a gene, amyA, that encodes a cytoplasmic a-amylase. The chromosome between amyA and flagellar region IIIb has now been investigated. The high level of DNA similarity between the E. coli and S. typhimurium sequences that exists in flagellar region IIIa and in amyA continues initially, with three genes of unknown function; in E. coli, there may be a fourth gene. The remainder of the region, up to the start of flagellar region IIIb, lacks any obvious open reading frames, scores poorly on an algorithm for coding probability, has a high A + T content, and is totally dissimilar in the two species. We conclude that it is non-coding. In E. coli this region extends for 2.7 kb and in S. typhimurium for 0.8 kb. These values are unusually large for prokaryotes, where the non-coding regions between operons are generally quite short. The data, which are discussed in the context of a hypothesized disruption of a contiguous ancestral flagellar region, may give new insight into the organization and evolution of the bacterial chromosome.