Showing papers in "Journal of Bacteriology in 1993"

Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells.

Abstract: A 31-kb fragment of the large virulence plasmid of Shigella flexneri is necessary for bacterial entry into epithelial cells in vitro. One locus of this fragment encodes the IpaA, -B, -C, and -D proteins, which are the dominant antigens of the humoral immune response during shigellosis. To address the role of the ipa genes, which are clustered in an operon, we constructed a selectable cassette that does not affect transcription of downstream genes and used this cassette to inactivate the ipaB, ipaC, and ipaD genes. Each of these nonpolar mutants was defective in entry and lysis of the phagocytic vacuole but was not impaired in adhesion to the cells. We showed that, like IpaB and IpaC, IpaD is secreted into the culture supernatant and that none of these proteins is necessary for secretion of the other two. This result differentiates the Ipa proteins, which direct the entry process, from the Mxi and Spa proteins, which direct secretion of the Ipa proteins. Moreover, lack of either IpaB or IpaD resulted in the release of larger amounts of the other Ipa polypeptides into the culture medium, which indicates that, in addition to their role in invasion, IpaB and IpaD are each involved in the maintenance of the association of the Ipa proteins with the bacterium.

Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon.

Abstract: An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug efflux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB.

Adhere today, here tomorrow: oral bacterial adherence.

Abstract: The oral cavity is home to a large and diverse population of microbes comprising over 350 taxa and in which 37 genera of bacteria are represented (37, 38). Coaggregation, or cellto-cell recognition of genetically distinct partner cell-types, has been observed with isolates from the 18 genera tested so far (25, 26); these 18 genera constitute the bacteria most frequently isolated from dental plaque. Like other saprophytic and pathogenic bacteria that inhabit or invade various human tissues, members of the oral flora possess specific cell surface-associated adherence proteins responsible for initiating colonization. These bacterial adhesins recognize protein, glycoprotein, or polysaccharide receptors on various oral surfaces, including other cell types. In dental plaque, the ability to attach to bacteria already anchored to hard or soft tissues may provide secondary colonizers with the same advantages enjoyed by primary colonizers. Essentially all oral bacteria possess surface molecules that foster some sort of cell-to-cell interaction (26); some constitutively synthesize a coterie of adhesins that permit a cell to participate simultaneously in multiple interactions with partner cell types (32). Other oral bacteria, such as the cariogenic mutans streptococci, synthesize extracellular glucans and a major surface protein antigen (14) that contribute to their ability to adhere to teeth (29). This kind of adherence has been extensively studied by many laboratories and appears to be distinct from coaggregation. In other ecosystems, there is surprisingly little or no evidence for coaggregation among resident bacteria. Juxtapositioning of genetically unrelated bacterial microcolonies in metabolic consortia performing anaerobic biodegradations (36, 49) is different from coaggregations which are characterized by direct and viability-independent cell-to-cell recognition. Bacterial predation and conjugation, the action of the pheromone system of Enterococcus faecalis (44), invertebrate animal intestinal tract microbes (5), and nutritional symbionts have some similarities to the highly specific mechanisms of oral bacterial recognition. Coaggregation may be intra-, interor multigeneric, and it is different from the interactions among clonal populations, for example rosettes among caulobacters. Generally, secondary colonizers synthesize protein adhesins that recognize receptors on primary colonizers such as the streptococci and actinomyces (30). This review will deal with the characteristics of adhesin and receptor molecules and the potential roles they play in dental plaque accretion. Equally important to adherence in developing oral microbial consortia are the nutritional relationships among plaque bacteria, which have been reviewed elsewhere (26) and will not be discussed further here.

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Abstract: Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.

Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon.

Abstract: Acetohydroxy acid synthase (AHAS) and isomeroreductase (IR) catalyze subsequent reactions in the flux of metabolites towards isoleucine, valine, leucine, and pantothenate. A 4,705-bp DNA fragment from Corynebacterium glutamicum known to code for AHAS and IR was sequenced and analyzed by Northern (RNA blot) analysis. As in other bacteria, the AHAS of this gram-positive organism is encoded by two genes, ilvB and ilvN. Gene disruption verified that these genes encode the single AHAS activity in C. glutamicum. The start of ilvB was determined by amino-terminal sequencing of a fusion peptide. By Northern analysis of the ilvBNC cluster, three in vivo transcripts of 3.9, 2.3, and 1.1 kb were identified, corresponding to ilvBNC, ilvNC, and ilvC messages, respectively. The ilvC transcript (encoding IR) was by far the most abundant one. With a clone from which the ilvB upstream regions had been deleted, only the ilvNC and ilvC transcripts were synthesized, and with a clone from which the ilvN upstream regions had been deleted, only the smallest ilvC transcript was formed. It is therefore concluded that in the ilv operon of C. glutamicum, three promoters are active. The amounts of the ilvBNC and ilvNC transcripts increased in response to the addition of alpha-ketobutyrate to the growth medium. This was correlated to an increase in specific AHAS activity, whereas IR activity was not increased because of the relatively large amount of the ilvC transcript present under all conditions assayed. Therefore, the steady-state level of the ilvBNC and ilvNC messages contributes significantly to the total activity of the single AHAS. The ilvC transcript of this operon, however, is regulated independently and present in a large excess, which is in accord with the constant IR activities determined.

The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels.

Abstract: The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene. This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low. After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E. coli K-12, E. coli B, and Salmonella typhimurium. To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6. DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110. This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E. coli strains, as confirmed by analysis of protein synthesis in minicells. The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.

Abstract: A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system. Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar frequencies. Examination of randomly selected transformants by biolistics showed that 15 to 40% were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

Abstract: A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.

Molecular cloning and characterization of acrA and acrE genes of Escherichia coli.

Abstract: The DNA fragment containing the acrA locus of the Escherichia coli chromosome has been cloned by using a complementation test. The nucleotide sequence indicates the presence of two open reading frames (ORFs). Sequence analysis suggests that the first ORF encodes a 397-residue lipoprotein with a 24-amino-acid signal peptide at its N terminus. One inactive allele of acrA from strain N43 was shown to contain an IS2 element inserted into this ORF. Therefore, this ORF was designated acrA. The second downstream ORF is predicted to encode a transmembrane protein of 1,049 amino acids and is named acrE. Genes acrA and acrE are probably located on the same operon, and both of their products are likely to affect drug susceptibilities observed in wild-type cells. The cellular localizations of these polypeptides have been analyzed by making acrA::TnphoA and acrE::TnphoA fusion proteins. Interestingly, AcrA and AcrE share 65 and 77% amino acid identity with two other E. coli polypeptides, EnvC and EnvD, respectively. Drug susceptibilities in one acrA mutant (N43) and one envCD mutant (PM61) have been determined and compared. Finally, the possible functions of these proteins are discussed.

Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp.

Abstract: Strains of Escherichia coli which lack detectable guanosine 39,59-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth. Images

Characterization of the ferrous iron uptake system of Escherichia coli.

Abstract: Escherichia coli has an iron(II) transport system (feo) which may make an important contribution to the iron supply of the cell under anaerobic conditions. Cloning and sequencing of the iron(II) transport genes revealed an open reading frame (feoA) possibly coding for a small protein with 75 amino acids and a membrane protein with 773 amino acids (feoB). The upstream region of feoAB contained a binding site for the regulatory protein Fur, which acts with iron(II) as a corepressor in all known iron transport systems of E. coli. In addition, a Fnr binding site was identified in the promoter region. The FeoB protein had an apparent molecular mass of 70 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was localized in the cytoplasmic membrane. The sequence revealed regions of homology to ATPases, which indicates that ferrous iron uptake may be ATP driven. FeoA or FeoB mutants could be complemented by clones with the feoA or feoB gene, respectively. Images

The sigma 54 bacterial enhancer-binding protein family: mechanism of action and phylogenetic relationship of their functional domains.

Abstract: Because of their ability to stimulate gene expression from distant sites and regardless of their orientation, transcriptional enhancer elements are a puzzling class of regulatory DNA sequences (42). Although enhancers are a common feature of eukaryotic and viral genes, the mechanism by which they communicate with promoters and stimulate gene expression is still not very well understood. However, our understanding of the mechanism of gene activation at a distance has been boosted by the study of naturally occurring enhancer-like elements (ELE) in prokaryotes. The ELE are palindromic or nearly palindromic nucleotide sequences normally found between 100 and 200 bp upstream of a certain class of promoters, and, like the eukaryotic enhancers, they still stimulate transcription when moved thousands of nucleotides upstream or downstream from the transcription start and regardless of their orientation (7, 47). The extensive genetic characterization of many bacterial ELE and the development of defined in vitro transcription systems has greatly facilitated the study of activation by enhancers, and now some of the molecular details of distal activation are well established (34). It has been demonstrated that the ELE are binding sites for regulatory proteins named bacterial enhancer-binding proteins (EBPs). The best-studied EBPs are NtrC and NifA, which stimulate the expression of genes required for nitrogen assimilation and nitrogen fixation, respectively, in a number of organisms (37). These proteins activate genes transcribed by the RNA polymerase holoenzyme containing the alternative sigma factor &F4 (EO(J4). The u54 system is widely represented in the alpha and gamma proteobacteria (previously called purple bacteria), and it has also been reported to be present in Bacillus subtilis (13, 14). Among other physiological processes controlled by C54 are nitrogen assimilation and fixation, dicarboxylic acid transport, hydrogen oxidation, alginate utilization, degradation of aromatic compounds, pilus formation and formate utilization (for reviews, see references 35 and 56). A common feature of all these processes is that they are not absolutely required for cell survival, and each is activated by a different EBP interacting with a distinctive ELE nucleotide sequence. In addition to its ability to be activated at a distance, the Er54 system has several other unique characteristics that are not common among bacterial holoenzymes. E&'4 recognizes promoter DNA with unusually close boxes, centered at

Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring.

Abstract: Immunoelectron microscopy was used to assess the effects of inhibitors of cell division on formation of the FtsZ ring in Escherichia coli. Induction of the cell division inhibitor SulA, a component of the SOS response, or the inhibitor MinCD, a component of the min system, blocked formation of the FtsZ ring and led to filamentation. Reversal of SulA inhibition by blocking protein synthesis in SulA-induced filaments led to a resumption of FtsZ ring formation and division. These results suggested that these inhibitors block cell division by preventing FtsZ localization into the ring structure. In addition, analysis of min mutants demonstrated that FtsZ ring formation was also associated with minicell formation, indicating that all septation events in E. coli involve the FtsZ ring.

Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli.

Abstract: A 7.8-kbp fragment of chromosomal DNA from a region controlling multiple antibiotic resistance (Mar) in Escherichia coli has been sequenced. Within the fragment is a potential divergent promoter region including marO, which contains two pairs of direct repeats, suggesting possible operator-regulatory sites. To the left of marO (region I) are one or two transcriptional units with three putative open reading frames (ORFs) encoding 64, 157, and 70 amino acids. To the right (region II) is a transcriptional unit containing three putative ORFs (ORF125/144, ORF129, and ORF72). Of six independent Mar mutants, four had mutations within the ORF encoding the first putative protein (ORF125/144) downstream of marO, including three different single-point mutations and an IS2 insertion. One of the other mutations occurred in marO (20-bp duplication), and the other occurred in a site in marO or ORF144 (a 1-bp change). All six mutations led to increased transcription of the region II transcript. High-copy-number plasmids containing marO and the adjacent ORF125/144 region from a wild-type source but not from a Mar mutant reduced the antibiotic resistance of a Mar mutant to levels comparable to those of wild-type cells. High-copy-number plasmids containing wild-type marO alone caused an increase in resistance to tetracycline, chloramphenicol, and norfloxacin in a wild-type strain. The nature of the Mar mutations and the results of the complementation studies suggest that ORF125/144 encodes a repressor (designated MarR) which acts at marO. The second ORF (ORF129), designated marA, would encode a protein, MarA, whose sequence shows strong similarity to those of a family of positive transcriptional regulators. A Tn5 insertion in marA inactivated the multiresistance phenotype of Mar mutants. The function of ORF72, designated marB, encoding the third putative protein in the operon, and that of other ORFs detected within the 7.8-kb fragment have not yet been determined.

The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein.

Abstract: The motile, alkalophilic, and extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila is positively photophobotactic. This response results in the accumulation of bacteria in light spots (E. Hustede, M. Liebergesell, and H. G. Schlegel, Photochem. Photobiol. 50:809-815, 1989; D. E. McRee, J. A. Tainer, T. E. Meyer, J. Van Beeumen, M. A. Cusanovich, and E. D. Getzoff, Proc. Natl. Acad. Sci. USA 86:6533-6537, 1989; also, this work). In this study, we demonstrated that E. halophila is also negatively phototactic. Video analysis of free-swimming bacteria and the formation of cell distribution patterns as a result of light-color boundaries in an anaerobic suspension of cells revealed the existence of a repellent response toward intense (but nondamaging) blue light. In the presence of saturating background photosynthetic light, an increase in the intensity of blue light induced directional switches, whereas a decrease in intense blue light gave rise to suppression of these reversals. To our knowledge, this is the first report of a true repellent response to light in a free-swimming eubacterium, since the blue light response in Escherichia coli and Salmonella typhimurium (B. L. Taylor and D. E. Koshland, Jr., J. Bacteriol. 123:557-569, 1975), which requires an extremely high light intensity, is unlikely to be a sensory process. The wavelength dependence of this negative photoresponse was determined with narrow band pass interference filters. It showed similarity to the absorption spectrum of the photoactive yellow protein from E. halophila. Images

Identification and molecular characterization of csrA, a pleiotropic gene from Escherichia coli that affects glycogen biosynthesis, gluconeogenesis, cell size, and surface properties.

Abstract: Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.

Protein-protein communication within the transcription apparatus.

Abstract: Despite the rapid expansion of catalogues of cis-acting transcription signals on DNA and of trans-acting transcription regulatory proteins found in both prokaryotes and eukaryotes, relatively little is known about precisely how these transcription signals and factors ultimately influence transcription. Recent progress in studies of the molecular anatomy of Escherichia coli RNA polymerase, however, has led to a breakthrough in our understanding of the molecular mechanisms underlying transcription regulation by transcription factors. The RNA polymerase holoenzyme of E. coli is composed of core enzyme with the subunit structure a2133' and one of the several species of v subunit which are involved in the specific recognition of promoters. The core enzyme is the basic machinery ofRNA synthesis: the catalytic site of RNA polytherization is located on the 1 subunit, while RNA polym rase binds to DNA nonspecifically via the 13' subunit. Subunit a links these two large subunits into the core enzyme complex (for reviews, see references 15, 17, and 43). The core enzyme is functionally differentiated into the various forms of holoenzyme by binding one of the different molecular species of cr subunit (for a review, see reference 10). Simple promoters are recognized by one or more of these holoenzymes, but some promoters require additional transcription factors for transcription initiation (1, 16, 17). An increasing amount of evidence indicates that interplay between RNA polymerase and transcription factors involves direct protein-protein contacts (19). Thus, the molecular architecture of the transcription apparatus for specific and accurate initiation differs in detail from promoter to promoter. Each transcription apparatus is responsible for transcription of only a set of genes. Since the number of core enzyme molecules is fixed at a constant level characteristic of the rate of cell growth, i.e., on average about 2,000 molecules per genome equivalent of DNA, the degree to which each of the approximately 4,000 genes on the E. coli chromosome is transcribed is primarily determined by the relative numbers of each kind of transcription apparatus with a particular promoter selectivity (16, 18). In this article, I will summarize the protein-protein communication between RNA polymerase and transcription factors from E. coli.

A new lipopeptide biosurfactant produced by Arthrobacter sp. strain MIS38.

Abstract: A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D- leucyl-D-leucyl-D-seryl-L-leucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-as paragyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25 degrees C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants. Images

Salicylate induction of antibiotic resistance in Escherichia coli: activation of the mar operon and a mar-independent pathway.

Abstract: Since the growth of wild-type Escherichia coli in salicylate results in a multiple antibiotic resistance phenotype similar to that of constitutive mutants (Mar) of the chromosomal mar locus, the effect of salicylate on the expression of the marRAB operon was investigated. The amount of RNA hybridizing with a mar-specific DNA probe was 5 to 10 times higher in wild-type cells grown with sodium salicylate (5.0 mM) than in untreated controls. Untreated Mar mutants had three to five times more mar-specific RNA than wild-type cells did. When a Mar mutant was treated with salicylate, a 30- to 50-fold increase of mar-specific RNA was seen. In wild-type cells bearing a mar promoter-lacZ fusion on the chromosome, salicylate increased beta-galactosidase activity by sixfold. Thus, salicylate induces transcription of the marRAB operon. Other inducers of phenotypic multiple antibiotic resistance, e.g., benzoate, salicyl alcohol, and acetaminophen, but not acetate, also increased transcription from the mar promoter but to a lesser extent than did salicylate. Both in wild-type and mar-deficient strains, growth in salicylate resulted in increased antibiotic resistance, decreased permeation of the outer membrane to cephaloridine, increased micF transcription, and decreased amounts of OmpF. However, the magnitude of these changes was generally greater in wild-type (mar-containing) cells. Thus, salicylate and other compounds can induce transcription of the mar operon and, presumably, give rise to multiple antibiotic resistance via this pathway. However, salicylate can also activate an unidentified, mar-independent pathway(s) which engenders multiple antibiotic resistance.

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Abstract: Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.

Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis.

Abstract: Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 1H-nuclear magnetic resonance analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts of L. lactis strains carrying Tn5276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.

Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.

Abstract: Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic conditions. Of the 30 cobalamin synthetic genes, 25 are clustered in one operon, cob, and are arranged in three groups, each group encoding enzymes for a biochemically distinct portion of the biosynthetic pathway. We have determined the DNA sequence for the promoter region and the proximal 17.1 kb of the cob operon. This sequence includes 20 translationally coupled genes that encode the enzymes involved in parts I and III of the cobalamin biosynthetic pathway. A comparison of these genes with the cobalamin synthetic genes from Pseudomonas denitrificans allows assignment of likely functions to 12 of the 20 sequenced Salmonella genes. Three additional Salmonella genes encode proteins likely to be involved in the transport of cobalt, a component of vitamin B12. However, not all Salmonella and Pseudomonas cobalamin synthetic genes have apparent homologs in the other species. These differences suggest that the cobalamin biosynthetic pathways differ between the two organisms. The evolution of these genes and their chromosomal positions is discussed.

A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells.

Abstract: The Salmonella typhimurium transcriptional regulators, PhoP/PhoQ, induce phoP-activated gene (pag) expression to promote virulence and intracellular survival within macrophages. This response to the macrophage intracellular environment is simulated by phoP/phoQ constitutive mutations (phenotype PhoPc) that increase the expression of pag genes and repress the synthesis of approximately 20 proteins encoded by phoP-repressed genes (prg genes) (S. I. Miller and J. J. Mekalanos, J. Bacteriol. 172:2485-2490, 1990). PhoPc bacteria are attenuated for mouse virulence, suggesting that prg genes are virulence genes. We now report the identification of five unlinked prg loci by use of the transposon TnphoA. In general, medium conditions (i.e., starvation) that activate pag expression repress prg expression. However, variable effects on the PhoP regulon were observed when bacteria were grown under different oxygen tensions (pag and prg genes) or exposed to low pH (prg genes), suggesting heterogenous control of the regulon. One prg locus, prgH, was demonstrated to contribute to mouse virulence by both the oral and the intraperitoneal routes. prgH was located at 59 min on the Salmonella chromosome, a region where other genes essential to invasion of epithelial cells are clustered. The prgH locus was highly linked to one invasion locus, hil (C.A. Lee, B.D. Jones, and S. Falkow, Proc. Natl. Acad. Sci. USA 89:1847-1851, 1992), although transcription of prgH was opposite that of the Tn5B50-encoded promoters that result in a hyperinvasive or hil phenotype. Both PrgH and PhoPc mutant S. typhimurium were found to be defective in induction of endocytosis by Madin-Darby canine kidney (MDCK) epithelial cells. The invasion defect of PrgH but not that of PhoPc mutant bacteria was complemented by plasmids containing prgH (hil) DNA. Therefore, two virulence properties of Salmonella species, induction of endocytosis by epithelial cells and survival within macrophages, are oppositely modulated by the PhoP/PhoQ virulence regulators.

Sequence of the gene coding for ammonia monooxygenase in Nitrosomonas europaea.

Abstract: Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

Unipolar localization and atpase activity of icsa, a shigella flexneri protein involved in intracellular movement

Abstract: Shigella flexneri uses elements of the host cell cytoskeleton to move within cells and from cell to cell. IcsA, an S. flexneri protein involved in this movement, was purified and studied in vitro. IcsA bound the radiolabelled ATP analog 39(29)-O-(4-benzoyl)benzoyl-ATP and hydrolyzed ATP. In addition, the surface localization of IcsA on both extracellular and intracellular shigellae was unipolar. Further, in HeLa cells infected with shigellae, IcsA antiserum labelled the actin tail throughout its length, thereby suggesting that IcsA interacts with elements within the tail. Localization of IcsA within the tail at a distance from the bacterium would require its secretion; we demonstrate here that in vitro IcsA is secreted into the culture supernatant in a cleaved form. Images

Is acetyl phosphate a global signal in Escherichia coli

Abstract: In this article we discuss recent evidence indicating that acetyl phosphate may be a global signal in Escherichia coli and related bacteria. We shall start by reviewing the evidence that acetyl phosphate is involved in signal transduction by a family of related phosphorylation-dependent switches known as the two-component systems. We shall also review the data indicating the mechanism by which acetyl phosphate affects the function of these switches. Finally, we shall review the factors affecting the intracellular concentration of acetyl phosphate in an attempt to deduce the significance of the signal provided by acetyl phosphate. We conclude by offering two suggestions for the role of acetyl phosphate as a potential global signal.

A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells.

Abstract: Enteropathogenic Escherichia coli (EPEC) is capable of attaching intimately to epithelial cells and effacing their microvilli. A chromosomal locus, eaeA (originally eae), is required for the intimate attachment aspect of this effect. We report the mapping of a region of the EPEC chromosome that is located immediately downstream of the eaeA gene and that is also necessary for intimate attachment. An isogenic in-frame deletion mutation in one of the open reading frames identified in this region was engineered. Because the resulting mutant, like an eaeA deletion mutant, is deficient in the ability to attach intimately to epithelial cells, the mutated gene is designated eaeB. Full activity is restored to the eaeB mutant when the cloned gene is reintroduced on a plasmid. The eaeB mutant remains capable of producing intimin, the product of the eaeA gene. No differences in the fractionation properties or electrophoretic mobility of intimin are apparent in the eaeB mutant. The product of the eaeB locus was identified by in vitro transcription-translation. The nucleotide sequence of the eaeB gene predicts a protein that contains a sequence motif common to several aminotransferase enzymes. These results indicate that the attaching and effacing effect is a complex phenotype dependent on a gene cluster present on the EPEC chromosome.

Bacterial endotoxins: extraordinary lipids that activate eucaryotic signal transduction.

Abstract: Over 100 years ago, Pfeiffer and Centanni described heat-stable, nonsecreted toxin(s) in gram-negative bacteria that caused fever and pathology when injected into animals (53). Unlike heat-labile protein toxins, such as diptheria and tetanus toxins, the cell-associated toxins of gram-negative bacteria (endotoxins) consisted of carbohydrates and lipids (36, 53) and are also termed lipopolysaccharides (LPS) (see Fig. 1). Uncertainties about the chemical nature of endotoxins lingered until 1983, when the structure of the lipid A anchor of LPS (see Fig. 1 and 2) was elucidated (36, 46, 53), and chemically synthesized lipid A was shown to possess the biological activities of endotoxins (36, 46, 53). The long delay in establishing the identity of the active component of endotoxins occurred because the structure of lipid A is complex (29, 36, 46) and differs from those of all other lipids (45). The approximate composition of lipid A was recognized 40 years ago (36, 53), but its biosynthesis and pharmacology could not be studied without a complete understanding of its covalent chemistry (46). In 1983, just as several laboratories were close to defining the structure of lipid A (54), my group characterized a novel glucosamine-derived phospholipid (58), previously observed in certain phosphatidylglycerol-deficient mutants of Escherichia coli (40). Together with Laurens Anderson and Kuni Takayama at the University of Wisconsin (58), we showed that this material, termed lipid X, was a 2,3-diacylglucosamine 1-phosphate (see Fig. 2). The discovery of a monosaccharide substructure of lipid A provided an important clue to elucidating the sites of acylation on lipid A (40, 54, 58) and the enzymatic pathway for lipid A biosynthesis (1, 14, 46, 50). The functional connection between lipid X accumulation and phosphatidylglycerol deficiency remains unknown (39, 40). In this minireview, I summarize current knowledge of the enzymatic synthesis of lipid A, most of which is derived from studies with E. coli extracts (46, 65). I also provide a brief overview of the mechanisms by which lipid A activates signal transduction in animal cells.

Stress response of Escherichia coli to elevated hydrostatic pressure.

Abstract: The response of exponentially growing cultures of Escherichia coli to abrupt shifts in hydrostatic pressure was studied A pressure upshift to 546 atm (55,304 kPa) of hydrostatic pressure profoundly perturbed cell division, nucleoid structure, and the total rate of protein synthesis The number of polypeptides synthesized at increased pressure was greatly reduced, and many proteins exhibited elevated rates of synthesis relative to total protein synthesis We designated the latter proteins pressure-induced proteins (PIPs) The PIP response was transient, with the largest induction occurring approximately 60 to 90 min postshift Fifty-five PIPs were identified Many of these proteins are also induced by heat shock or cold shock The PIP demonstrating the greatest pressure induction was a basic protein of 156 kDa High pressure inhibits growth but does not inhibit the synthesis of stringently controlled proteins Cold shock is the only additional signal which has been found to elicit this type of response These data indicate that elevated pressure induces a unique stress response in E coli, the further characterization of which could be useful in delineating its inhibitory nature

Stress-induced activation of the sigma B transcription factor of Bacillus subtilis.

Abstract: The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals. On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions. Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response. After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression. Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth. Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells. However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus.