Abstract: A second high-frequency switching system was identified in selected pathogenic strains in the dimorphic yeast Candida albicans. In the characterized strain WO-1, cells switched heritably, reversibly, and at a high frequency (approximately 10(-2] between two phenotypes readily distinguishable by the size, shape, and color of colonies formed on agar at 25 degrees C. In this system, referred to as the "white-opaque transition," cells formed either "white" hemispherical colonies, which were similar to the ones formed by standard laboratory strains of C. albicans, or "opaque" colonies, which were larger, flatter, and grey. At least three other heritable colony phenotypes were generated by WO-1 and included one irregular-wrinkle and two fuzzy colony phenotypes. The basis of the white-opaque transition appears to be a fundamental difference in cellular morphology. White cells were similar in shape, size, and budding pattern to cells of common laboratory strains. In dramatic contrast, opaque cells were bean shaped and exhibited three times the volume and twice the mass of white cells, even though these alternative phenotypes contained the same amount of DNA and a single nucleus in the log phase. In addition to differences in morphology, white and opaque cells differed in their generation time, in their sensitivity to low and high temperatures, and in their capacity to form hypae. The possible molecular mechanisms involved in high-frequency switching in the white-opaque transition are considered.

Abstract: The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.

Abstract: Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.

Abstract: The role of motility in the colonization of potato roots by Pseudomonas bacteria was studied. Four Tn5-induced flagella-less mutants of the plant-growth-stimulating P. fluorescens WCS374 appeared to be impaired in their ability to colonize growing potato roots.

Abstract: Shiga-like toxin is an iron-regulated cytotoxin quite similar to Shiga toxin from Shigella dysenteriae 1. The structural genes for Shiga-like toxin in Escherichia coli (sltA and sltB) appear to be transcribed as an operon from a promoter upstream of sltA. We used a gene fusion between the promoter and proximal portion of sltA with the gene for bacterial alkaline phosphatase to assess the regulation of toxin expression. Growth in low-iron conditions resulted in a 13- to 16-fold increase in alkaline phosphatase activity. In the presence of a null mutation in the fur locus, however, alkaline phosphatase activity was constitutively high regardless of the iron concentration. These data indicate negative regulation of the slt operon by the fur gene product. We used deletion analysis of the region upstream of the gene fusion to localize the promoter of the slt operon and to show that a region of DNA between the -35 and -10 boxes is necessary for iron regulation of slt expression. In this region, there is a 21-base-pair dyad repeat that is homologous to similar dyads in the promoter regions of three other fur-regulated genes. This region of dyad symmetry may represent an operator binding site for the Fur protein in the presence of iron.

Abstract: The pelB gene encodes pectate lyase B, one of three pectate lyases identified in Erwinia carotovora EC. Pectate lyase B was purified from Escherichia coli containing the pelB gene on a recombinant plasmid. The activity of the protein was optimal at a pH of 8.3. The amino acid composition, N-terminal amino acid sequence, and C-terminal peptide sequence were determined and compared with the polypeptide sequence deduced from the DNA sequence of pelB. Purified pectate lyase B started at amino acid 23 of the predicted sequence, suggesting that a 22-amino-acid leader peptide had been removed. Pectate lyase B of E. carotovora EC and pectate lyase B of E. chrysanthemi EC16 contain 352 and 353 amino acids, respectively (N. T. Keen, S. Tanaki, W. Belser, D. Dahlbeck, and B. Staskawicz, J. Bacteriol. 168:595-606, 1986). The two proteins are 72% homologous on the basis of DNA sequence data, and 75% of the amino acids are identical.

Abstract: The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 genes encoding enzymes in 650 isolates of eight serogroups (A, B, C, W135, X, Y, Z, and 29E) and 38 nonserogroupable isolates. A total of 331 distinctive multilocus genotypes (electrophoretic types, ETs) was identified, among which mean genetic diversity per locus (H = 0.547) was greater than in Escherichia coli and other bacterial species thus far studied. The intercontinental distribution of some ETs and the recovery of organisms of identical genotype over periods of many years strongly suggest that the genetic structure of N. meningitidis is basically clonal as a consequence of low rates of recombination of chromosomal genes. Variation among strains in serogroup, serotype, and the electrophoretic pattern of the major outer membrane proteins has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at genetic distances greater than 0.50. Genetic diversity among ETs of isolates of the same serogroup was, on average, 84% of that in the total sample. Clones of serogroup A were unusual in being genotypically less heterogeneous than those of other serogroups and in forming a single phylogenetic group. Isolates of the same serotype or outer membrane protein pattern were also highly heterogeneous; on average, 87 and 97%, respectively, of the total species diversity was represented by ETs of the same serotype or outer membrane protein.

Abstract: lon mutants of Escherichia coli K-12 are defective in an ATP-dependent protease, are UV sensitive, and overproduce the capsular polysaccharide colanic acid. Six structural genes needed for capsular polysaccharide synthesis (cps) are transcriptionally regulated by lon as well as by three other regulatory genes, rcsA, -B, and -C (S. Gottesman, P. Trisler, and A. S. Torres-Cabassa, J. Bacteriol. 162:1111-1119, 1985). We have cloned rcsA, the gene for a positive regulator of capsule synthesis, onto multicopy plasmids and defined the gene by both insertions and deletions. The product of rcsA has been identified as an unstable protein of 27 kilodaltons. RcsA has a half-life of 5 min in lon+ cells and one of 20 min in lon cells. The availability of RcsA is the limiting factor for capsule synthesis; doubling the gene dosage of rcsA+ significantly increases expression of cps genes. Our results are consistent with a model in which the presence of a lon mutation increases the synthesis of capsular polysaccharide via stabilization of RcsA.

Abstract: A chromosomal region of Escherichia coli contiguous to the fabE gene at 71 min on the chromosomal map contains multiple genes that are responsible for determination of the rod shape and sensitivity to the amidinopenicillin mecillinam. The so-called mre region was cloned and analyzed by complementation of two closely related but distinct E. coli mutants characterized, respectively, by the mutations mre-129 and mre-678, that showed a rounded to irregular cell shape and altered sensitivities to mecillinam; the mre-129 mutant was supersensitive to mecillinam at 30 degrees C, but the mre-678 mutant was resistant. The mre-678 mutation also caused simultaneous overproduction of penicillin-binding proteins 1Bs and 3. A chromosomal region of the wild-type DNA containing the total mre region and the fabE gene was first cloned on a lambda phage; a 7-kilobase (kb) fragment containing the whole mre region, but not the fabE gene, was then recloned on a mini F plasmid, pLG339; and finally, a 2.8-kb fragment complementing only mre-129 was also cloned on this low-copy-number plasmid. The whole 7-kb fragment was required for complementing the mre-678 mutant phenotypes. Fragments containing fabE but not the mre-129 region could be cloned on a high-copy-number plasmid. Southern blot hybridization indicated that the mre-678 mutant had a large deletion of 5.25 kb in its DNA, covering at least part of the mre-129 gene.

Abstract: Plasmid pJP4 of Alcaligenes eutrophus JMP134 contains all genes for the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D). Five of these genes, tfdB, tfdC, tfdD, tfdE, and tfdF, have recently been localized and cloned (R. H. Don, A. J. Weightman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). Gene tfdA, which codes for the 2,4-D monooxygenase, has now been found by mutagenesis with transposon Tn5. A 3-kilobase fragment of pJP4 cloned in a broad-host-range vector could complement the 2,4-D-negative phenotype of two mutants which lacked 2,4-D monooxygenase activity. The cloned tfdA gene was also transferred to A. eutrophus JMP222, which is a cured derivative of JMP134. The recombinant strain could utilize phenoxyacetic acid as a sole source of carbon and energy. Pseudomonas sp. strain B13, containing the cloned tfdA, was able to degrade phenoxyacetic acid and 4-chlorophenoxyacetic acid. Gene tfdA was subcloned and analyzed by deletions. Expression of 2,4-D monooxygenase in Escherichia coli containing a 1.4-kilobase subfragment was demonstrated by radioisotopic enzyme assay, and a protein of 32,000-dalton molecular mass was detected by labeling experiments. A 2-kilobase subfragment containing tfdA has been sequenced. Sequence analysis revealed an open reading frame of 861 bases which was identified as the coding region of tfdA by insertion mutagenesis.

Abstract: We report the identification and cloning of an ntrA-like (glnF rpoN) gene of Rhizobium meliloti and show that the R. meliloti ntrA product (NtrA) is required for C4-dicarboxylate transport as well as for nitrate assimilation and symbiotic nitrogen fixation. DNA sequence analysis showed that R. meliloti NtrA is 38% homologous with Klebsiella pneumoniae NtrA. Subcloning and complementation analysis suggested that the R. meliloti ntrA promoter lies within 125 base pairs of the initiation codon and may be constitutively expressed.

Abstract: DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS) Images

Abstract: The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.

Abstract: Pertussis toxin, the major virulence factor of Bordetella pertussis, is not produced by the closely related species Bordetella parapertussis and Bordetella bronchiseptica. It is shown here that these two species possess but do not express the complete toxin operon. Nucleotide sequencing of an EcoRI fragment of 5 kilobases comprising the regions homologous to the pertussis toxin genes shows that in this region, B. parapertussis and B. bronchiseptica are 98.5% and 96% homologous, respectively, to B. pertussis. The changes (mostly base pair substitutions) in many cases are identical in B. parapertussis and B. bronchiseptica, suggesting that these two species derive from a common ancestor. Many of the mutations common to B. parapertussis and B. bronchiseptica involve the promoter region, which becomes very inefficient. The S1 subunits of both species, when expressed in Escherichia coli, have the same ADP-ribosylating activity as the S1 subunit from B. pertussis, indicating that the mutations in the S1 gene described here do not affect its function.

Abstract: Restriction fragment length polymorphisms in the ribosomal DNA (rDNA) have been shown to be a useful criterion for distinguishing among various isolates of Candida albicans. In a sample of 12 clinical isolates, we found six different classes based on variations in the fragments produced from genomic DNA by EcoRI and visualized after Southern transfer by being probed with a plasmid containing Saccharomyces cerevisiae rDNA. Some of the classes appeared to be heterozygous at the rDNA locus. Similar digestion of other Candida species showed that each could be identified on the basis of its restriction patterns. Since these are highly reiterated genes, the differences were apparent on ethidium bromide-stained gels; Southern transfers were not necessary. EcoRI restriction maps of the rDNA of C. albicans, C. stellatoidea, C. tropicalis, and C. guilliermondii were determined.

Abstract: Secreted hemolysins were extremely common among clinical isolates of Proteus mirabilis, Proteus vulgaris, and Morganella morganii, and hemolytic activity was either cell associated or cell free. Southern hybridization of total DNA from hemolytic isolates to cloned regions of the Escherichia coli alpha-hemolysin (hly) determinant showed clear but incomplete homology between genes encoding production of hemolysins in the four species. One of the two E. coli secretion genes, hlyD, hybridized only with DNA from P. vulgaris and M. morganii, which produced cell-free hemolysis, but not with that from P. mirabilis, which showed only cell-associated activity. Molecular cloning of the genetic determinants of cell-free hemolytic activity from P. vulgaris and M. morganii chromosomal DNA allowed their functional analysis via inactivation with the transposons Tn1000 and Tn5. Both hemolysin determinants were about 7.5 kilobase pairs and comprised contiguous regions directing regulation, synthesis, and specific secretion out of the cell. Transposon mutations which eliminated secretion of the Proteus and Morganella hemolysins could be complemented specifically by the E. coli hemolysin secretion genes hlyB or hlyD. Alignment of the physically and functionally defined hly determinants from P. vulgaris and M. morganii with that of the E. coli alpha-hemolysin confirmed a close genetic relationship but also indicated extensive evolutionary divergence.

Abstract: The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.

Abstract: Transcriptional regulation of alginate biosynthesis by Pseudomonas aeruginosa was studied. A DNA region complementing the alg-5 mutation within the alginate gene cluster was found by RNA-DNA dot blot and Northern hybridization to be transcriptionally activated in mucoid P. aeruginosa. This region was subcloned as a 3.2-kilobase BglII-ClaI DNA fragment on the broad-host-range controlled transcription vector pMMB24, and gene products were analyzed by expression from the tac promoter. A 48-kilodalton polypeptide was detected in extracts of P. aeruginosa and 35S-labeled Escherichia coli maxicells. By using the same expression system, GDPmannose dehydrogenase activity was detected in both P. aeruginosa and E. coli. Thus, gene algD coding for this enzyme was found to be present in the transcriptionally active DNA area. Insertion of the xylE gene within the BglII-ClaI fragment disrupted the induction of the 48-kilodalton polypeptide, GDPmannose dehydrogenase activity, and alg-5 complementing ability. With the algD-xylE transcription fusion, activation of algD gene expression was shown to occur in mucoid P. aeruginosa of different origins. In addition, regulation of the algD promoter activity was demonstrated to be mediated by a diffusible factor.

Abstract: It is now well recognized that the outer membranes of gram-negative bacteria serve as molecular sieves which permit the passage of small hydrophilic molecules of sizes below a given cutoff, i.e., the exclusion limit (21, 27). This sieving property, which permits uptake of small substrate molecules but excludes potentially harmful enzymes and other large hydrophilic molecules, is due to a class of proteins called porins (21). Porins form trans-outermembrane, water-filled channels, the dimensions of which determine the exclusion limit. An indication of their importance in bacteria is the observaton that Spirochaeta aurantia, an organism which apparently lacks lipopolysaccharide in its outer membrane (also called its outer sheath) (A. Kropinski and E. P. Greenberg, personal communication), nevertheless contains a major porin protein (19) whose properties are closely related to those of, e.g., Escherichia coli (7). Porins have now been identified and characterized for 32 species of bacteria (R. E. W. Hancock, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, in

Abstract: I investigated the significance of the intracellular accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and of the coordinated decrease in the GTP pool for initiating morphological and physiological differentiation of Streptomyces griseus, a streptomycin-producing strain. In solid cultures, aerial mycelium formation was severely suppressed by the presence of excess nutrients. However, decoyinine, a specific inhibitor of GMP synthetase, enabled the cells to develop aerial mycelia in the suppressed cultures at concentrations which only partially inhibited growth. A factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone) added exogenously had no such effect. Decoyinine was also effective in initiating the formation of submerged spores in liquid culture. The ability to produce streptomycin did not increase but decreased drastically on the addition of decoyinine. This sharp decrease in streptomycin production was accompanied by a decrease in intracellular accumulation of ppGpp. A relaxed (rel) mutant was found among 25 thiopeptin-resistant isolates which developed spontaneously. The rel mutant had a severely reduced ability to accumulate ppGpp during a nutritional shift-down and also during postexponential growth and showed a less extensive decrease in the GTP pool than that in the rel+ parental strain. The rel mutant failed to induce the enzymes amidinotransferase and streptomycin kinase, which are essential for the biosynthesis of streptomycin. The abilities to form aerial mycelia and submerged spores were still retained, but the amounts were less, and for both the onset of development was markedly delayed. The decreased ability to produced submerged spores was largely restored by the addition of decoyinine. This was accompanied by an extensive GTP pool decrease. The rel mutant produced A factor normally, indicating that synthesis of A factor is controlled neither by ppGpp nor by GTP. Conversely, a mutant defective in A-factor synthesis accumulated as much ppGpp as did the parental strain. It was concluded that morphological differentiation of S. griseus results from a decrease in the pool of GTP, whereas physiological differentiation results from a more direct function of the rel gene product (ppGpp). It is also suggested that A factor may render the cell sensitive to receive and respond to the specified signal molecules, presumably ppGpp (for physiological differentiation) or GTP (for morphological differentiation).

Abstract: Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.

Abstract: Carbon-phosphorus bond cleavage activity, found in bacteria that utilize alkyl- and phenylphosphonic acids, has not yet been obtained in a cell-free system. Given this constraint, a systematic examination of in vivo C-P lyase activity has been conducted to develop insight into the C-P cleavage reaction. Six bacterial strains were obtained by enrichment culture, identified, and characterized with respect to their phosphonic acid substrate specificity. One isolate, Agrobacterium radiobacter, was shown to cleave the carbon-phosphorus bond of a wide range of substrates, including fosfomycin, glyphosate, and dialkyl phosphinic acids. Furthermore, this organism processed vinyl-, propenyl-, and propynylphosphonic acids, a previously uninvestigated group, to ethylene, propene, and propyne, respectively. A determination of product stoichiometries revealed that both C-P bonds of dimethylphosphinic acid are cleaved quantitatively to methane and, furthermore, that the extent of C-P bond cleavage correlated linearly with the specific growth rate for a range of substrates. The broad substrate specificity of Agrobacterium C-P lyase and the comprehensive characterization of the in vivo activity make this an attractive system for further biochemical and mechanistic experiments. In addition, the failure to observe the activity in a group of gram-positive bacteria holds open the possibility that a periplasmic component may be required for in vivo expression of C-P lyase activity.

Abstract: Strains missing several genes required for chemotaxis toward amino acids, peptides, and certain sugars were tethered and their rotational behavior was analyzed. Null strains (called gutted) were deleted for genes that code for the transducers Tsr, Tar, Tap, and Trg and for the cytoplasmic proteins CheA, CheW, CheR, CheB, CheY, and CheZ. Motor switch components were wild type, flaAII(cheC), or flaBII(cheV). Gutted cells with wild-type motors spun exclusively counterclockwise, while those with mutant motors changed their directions of rotation. CheY reduced the bias (the fraction of time that cells spun counterclockwise) in either case. CheZ offset the effect of CheY to an extent that varied with switch allele but did not change the bias when tested alone. Transducers also increased the bias in the presence of CheY but not when tested alone. However, cells containing transducers and CheY failed to respond to attractants or repellents normally detected in the periplasm. This sensitivity was restored by addition of CheA and CheW. Thus, CheY both enhances clockwise rotation and couples the transducers to the flagella. CheZ acts, at the level of the motor, as a CheY antagonist. CheA or CheW or both are required to complete the signal pathway. A model is presented that explains these results and is consistent with other data found in the literature.

Abstract: The structural organization and regulation of the genes involved in short-chain fatty acid degradation in Escherichia coli, referred to as the ato system, have been studied by a combination of classic genetic and recombinant DNA techniques. A plasmid containing a 6.2-kilobase region of the E. coli chromosome was able to complement mutations in the ato structural genes, atoA (acetyl-coenzyme A [CoA]:acetoacetyl [AA]-CoA transferase) and atoB (thiolase II), as well as mutations in the ato regulatory locus, atoC. Complementation studies performed with mutants defective in acetyl-CoA:AA-CoA transferase suggest that two loci, atoD and atoA, are required for the expression of functional AA-CoA transferase. The ato gene products were identified by in vitro transcription and translation and maxicell analysis as proteins of 48, 26.5, 26, and 42 kilodaltons for atoC, atoD, atoA, and atoB, respectively. In vitro and insertional mutagenesis of the ato hybrid plasmid indicated that the ato structural genes were arranged as an operon, with the order of transcription atoD-atoA-atoB. Although transcribed in the same direction as the atoDAB operon, the atoC gene appeared to use a promoter which was distinct from that used by the atoDAB operon. A delta atoC plasmid expressed the atoD, atoA, and atoB gene products only in strains containing a functional atoC gene. Although the exact mechanism of control was not evident from these studies, the data suggest that the atoC gene product is an activator which is required for the synthesis or activation of the atoDAB-encoded enzymes.

Abstract: Expression of fimA, the structural gene for type 1 fimbriae of Escherichia coli, is phase variable. Significant homologies were identified between the recombinases which control fimbrial phase variation, FimB and FimE, and the integrase class of site-specific recombinases. Normal expression of fimA was shown to require the integration host factor (IHF). Mutations in either the himA-or the himD (hip) gene, which encode the alpha and beta subunits of IHF, respectively, prevented phase variation and locked expression of fimA in either the "on" or "off" phase. In addition, both himA and himD lesions caused a sevenfold reduction in expression of a phi(fimA-lacZ) operon fusion in strains in which fimA was locked in the on phase. Thus, IHF plays a dual role in controlling fimA expression: it is required both for inversion of the fimA control region and for efficient expression from the fimA promoter. A mechanism by which IHF may exert control over fimA expression is discussed.

Abstract: We have previously described an assay for the attachment of Rhizobium bacteria to pea root hair tips (cap formation) which was used as a model to study the attachment step in the nodulation process. Under all conditions tested, a positive correlation was observed between the percentage of fibrillated cells and the ability of these bacteria to form caps and to adhere to glass, suggesting that fibrils play a role in the attachment of Rhizobium leguminosarum to pea root hair tips and to glass (G. Smit, J. W. KiJne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986). In the present paper the chemical and functional characterization of the fibrils of R. leguminosarum is described. Characterization of purified fibrils by infrared spectroscopy and cellulase treatment followed by thin-layer chromatography showed that the fibrils are composed of cellulose. Purified cellulose fibrils, as well as commercial cellulose, inhibited cap formation when present during the attachment assay. Incubation of the bacteria with purified cellulase just before the attachment assay strongly inhibited cap formation, indicating that the fibrils are directly involved in the attachment process. TnS-induced fibril-overproducing mutants showed a greatly increased ability to form caps, whereas TnS-induced fibrilnegative mutants lost this ability. None of these TnS insertions appeared to be located on the Sym plasmid. Both types of mutants showed normal nodulation properties, indicating that cellulose fibrils are not a prerequisite for successful nodulation under the conditions used. The ability of the fibril-negative mutants to attach to glass was not affected by the mutations, indicating that attachment to pea root hair tips and attachment to glass are (partly) based on different mechanisms. However, growth of the rhizobia under low Ca2+ conditions strongly reduced attachment to glass and also prevented cap formation, although it had no negative effect on fibril synthesis. This phenomenon was found for several Rhizobium spp. It was concluded that both cellulose fibrils and a Ca2 -dependent adhesin(s) are involved in the attachment of R. leguminosarum to pea root hair tips. A model for cap formation as a two-step process is discussed.

Abstract: Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).

Abstract: Deletion of sigE, the structural gene for the sporulation-induced RNA polymerase sigma factor, sigma E, prevented endospore formation by Bacillus subtilis. The effects of integration of plasmids into the sigE region of the chromosome and the use of complementation analyses demonstrated that sigE is part of an operon that includes a promoter-proximal gene, spoIIGA, that is essential for sporulation. Gene fusions to the promoter of this operon, spoIIG, demonstrated that transcription from this promoter is induced at the beginning of sporulation and is dependent on several spoO genes.

Abstract: Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.