Showing papers in "Journal of Bacteriology in 1994"

Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators.

Abstract: It has long been appreciated that certain groups of bacteria exhibit cooperative behavioral patterns. For example, feeding and sporulation of both myxobacteria and actinomycetes seem optimized for large populations of cells behaving almost as a single multicellular organism. The swarming motility of microorganisms such as Vibrio parahaemolyticus and Proteus mirabilis provides another excellent example of multicellular behavior among bacteria (2). Intercellular communication likewise has been appreciated for several years in Vibrio fischeri, Myxococcus xanthus, Bacillus subtilis, Streptomyces spp., the eukaryotic slime mold Dictyostelium discoideum, and other species (44). Here we first review how the marine luminescent bacterium V. fischeri uses the LuxR and LuxI proteins for intercellular communication and then describe a newly discovered family of LuxR and LuxI homologs in diverse bacterial species.

Biofilms, the customized microniche.

Abstract: At its 1993 annual meeting, the American Society for Microbiology deemed the biofilm mode of growth to be a concept worthy of an extraordinary 4-day colloquium consisting of 52 lectures from invited speakers. Perhaps, immediately following this herculean exercise, it is germane to consider the available evidence for the importance and uniqueness of this mode of growth and to assess the consequences of this concept in modern microbiology. It is, clearly, timely to ask the question "What are the essential differences between a planktonic cell growing in the conventional batch culture and a cell of the same species growing in a natural multispecies biofilm?" This question can be asked with some urgency, because whether we intended it or not, microbiologists have actually extrapolated between laboratory cultures and real ecosystems for hundreds of years and, lately, these extrapolations appear to have become increasingly strained and inaccurate.

The winds of (evolutionary) change: breathing new life into microbiology.

Abstract: To date, over 1500 prokaryotes have been characterized by small subunit rRNA sequencing and molecular phylogeny has had an equally profound effect on our understanding of relationship among eukaryotic microorganisms. The universal phylogenetic tree readily shows however how artificial the strong distinction between the eukaryote and prokaryotes has become. The split between the Archaea and the Bacteria is now recognized as the primary phylogenetic division and that the Eucarya have branched from the same side of the tree as the Archaea. Both prokaryotic domains would seem to be of thermophilic origin suggesting that life arose in a very warm environment. Among the Archaea, all of the Crenarchaeota cultured to date are thermophiles, and the deepest euryarchaeal branchings are represented exclusively by thermophiles. Among the Bacteria, the deepest known branchings are again represented exclusively by thermophiles, and thermophilia is widely scattered throughout the domain. The Archaea comprise a small number of quite disparate phenotypes that grow in unusual niches. All are obligate or facultative anaerobes. All cultured crenarchaeotes are thermophilic, some even growing optimally above the normal boiling temperature of water. The Archaeoglobales are sulfate reducers growing at high temperatures. The extreme halophiles grow only in highly saline environments. The methanogensmore » are confined to a variety of anaerobic niches, often thermophilic. The Bacteria, on the other hand, are notable as being the source of life`s photosynthetic capacity. Five kingdoms of bacteria contain photosynthetic species; and each of the five manifests a distinct type of (chlorophyll-based) photosynthesis.« less

A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite.

Abstract: Conjugal transfer of Agrobacterium octopine-type Ti plasmids is activated by octopine, a metabolite released from plant tumors. Octopine causes conjugal donors to secrete a pheromone, Agrobacterium autoinducer (AAI), and exogenous AAI further stimulates conjugation. The putative AAI synthase and an AAI-responsive transcriptional regulator were found to be encoded by the Ti plasmid traI and traR genes, respectively, and the expression of traR was induced by octopine. The octopine-type traR gene product is highly homologous to the TraR protein recently characterized from a nopaline-type Ti plasmid. TraR and TraI are homologous to the LuxR and LuxI regulatory proteins of Vibrio fischeri, and AAI is similar in structure to the diffusable V. fischeri autoinducer, the inducing ligand of LuxR. TraR activated target genes in the presence of AAI and also activated traR and traI themselves, creating two positive-feedback loops. TraR-AAI-mediated activation in wild-type Agrobacterium strains was dramatically enhanced by culturing on solid media, suggesting a possible role in cell density sensing.

Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria.

Abstract: Comparative 16S rRNA sequencing was used to evaluate phylogenetic relationships among selected strains of ammonia- and nitrite-oxidizing bacteria. All characterized strains were shown to be affiliated with the proteobacteria. The study extended recent 16S rRNA-based studies of phylogenetic diversity among nitrifiers by the comparison of eight strains of the genus Nitrobacter and representatives of the genera Nitrospira and Nitrospina. The later genera were shown to be affiliated with the delta subdivision of the proteobacteria but did not share a specific relationship to each other or to other members of the delta subdivision. All characterized Nitrobacter strains constituted a closely related assemblage within the alpha subdivision of the proteobacteria. As previously observed, all ammonia-oxidizing genera except Nitrosococcus oceanus constitute a monophyletic assemblage within the beta subdivision of the proteobacteria. Errors in the 16S rRNA sequences for two strains previously deposited in the databases by other investigators (Nitrosolobus multiformis C-71 and Nitrospira briensis C-128) were corrected. Consideration of physiology and phylogenetic distribution suggested that nitrite-oxidizing bacteria of the alpha and gamma subdivisions are derived from immediate photosynthetic ancestry. Each nitrifier retains the general structural features of the specific ancestor9s photosynthetic membrane complex. Thus, the nitrifiers, as a group, apparently are not derived from an ancestral nitrifying phenotype.

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

Abstract: A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Abstract: Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.

The superfamily of heme-copper respiratory oxidases.

Abstract: It has been estimated that about 90% of the reduction of molecular oxygen in the biosphere is catalyzed by respiratory oxidases (95). Much of this is accomplished by a wide variety of bacterial oxidases. It has long been recognized that bacterial respiratory systems are branched, having a number of distinct terminal oxidases, rather than the single cytochrome c oxidase present in most eukaryotic mitochondrial systems. This multiplicity of oxidases in bacteria has long been a source of confusion, both in terms of the nature of the enzymes themselves and in terms of the rationale for such complex systems. During the past several years, it has been recognized that most of the bacterial oxidases, despite differences in their substrates (quinol versus cytochrome c), oxygen affinities, and heme types and metal compositions, are closely related members of a single superfamily called the heme-copper oxidase superfamily. A large number of enzymes which just a few years ago were considered to be totally unrelated are now known to be close relatives. This recognition has provided considerable order to a chaotic field, and the research that has been stimulated in this field has generated new insights into heme biosynthesis and the evolution of respiratory systems. The heme-copper oxidase superfamily also includes the eukaryotic mitochondrial oxidases. Much of the impetus for pursuing the study of the bacterial oxidases has been to exploit the experimental advantages offered by the prokaryotic systems to learn about the structures and the functional mechanisms common to the members of the superfamily. The mitochondrial cytochrome c oxidase (primarily the bovine oxidase) has long been the object of intensive study (for reviews, see references 6, 10, 17, 20, 42, 59, and 86). This enzyme catalyzes the four-electron reduction of molecular oxygen to two molecules of water and utilizes the free energy available from this reaction to pump protons (one per electron) across the mitochondrial inner membrane, thus generating a transmembrane proton electrochemical gradient, or proton motive force (118). Although they typically contain only 3 or 4 subunits, in contrast to the 13-subunit mammalian enzyme, the bacterial oxidases catalyze the reduction of dioxygen and pump protons as efficiently as the mitochondrial oxidases do (39). Studies of several of the bacterial oxidases have made a very significant contribution to our knowledge of

Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses.

Abstract: The invasive properties of Azoarcus sp. strain BH72, an endorhizospheric isolate of Kallar grass, on gnotobiotically grown seedlings of Oryza sativa IR36 and Leptochloa fusca (L.) Kunth were studied. Additionally, Azoarcus spp. were localized in roots of field-grown Kallar grass. To facilitate localization and to assure identity of bacteria, genetically engineered microorganisms expressing beta-glucuronidase were also used as inocula. beta-Glucuronidase staining indicated that the apical region of the root behind the meristem was the most intensively colonized. Light and electron microscopy showed that strain BH72 penetrated the rhizoplane preferentially in the zones of elongation and differentiation and colonized the root interior inter- and intracellularly. In addition to the root cortex, stelar tissue was also colonized; bacteria were found in the xylem. No evidence was obtained that Azoarcus spp. could reside in living plant cells; rather, plant cells were apparently destroyed after bacteria had penetrated the cell wall. A common pathogenicity test on tobacco leaves provided no evidence that representative strains of Azoarcus spp. are phytopathogenic. Compared with the control, inoculation with strain BH72 significantly promoted growth of rice seedlings. This effect was reversed when the plant medium was supplemented with malate (0.2 g/liter). N2 fixation was apparently not involved, because the same response was obtained with a nifK mutant of strain BH72, which has a Nif- phenotype. Also, Western blot (immunoblot) analysis of protein extracts from rice seedlings gave no indication that nitrogenase was present. PCR and Western immunoblotting, using primers specific for eubacteria and antibodies recognizing type-specific antigens, respectively, indicated that strain BH72 could colonize rice plants systemically, probably mediated by longitudinal spreading through vessels.

Guanine nucleotide-dependent assembly of FtsZ into filaments.

Abstract: FtsZ is an essential cell division protein that is localized to the leading edge of the bacterial septum in a cytokinetic ring. It contains the tubulin signature motif and is a GTP binding protein with a GTPase activity. Further comparison of FtsZ with eukaryotic tubulins revealed some additional sequence similarities, perhaps indicating a similar GTP binding site. Examination of FtsZ incubated in vitro by electron microscopy revealed a guanine nucleotide-dependent assembly into protein filaments, supporting the hypothesis that the FtsZ ring is formed through self-assembly. FtsZ3, which is unable to bind GTP, does not polymerize, whereas FtsZ2, which binds GTP but is deficient in GTP hydrolysis, is capable of polymerization.

spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis.

Abstract: The spo0J gene of Bacillus subtilis is required for the initiation of sporulation. We show that the sporulation defect caused by null mutations in spo0J is suppressed by a null mutation in the gene located directly upstream from spo0J, soj (suppressor of spo0J). These results indicate that Soj inhibits the initiation of sporulation and that Spo0J antagonizes that inhibition. Further genetic experiments indicated that Soj ultimately affects sporulation by inhibiting the activation (phosphorylation) of the developmental transcription factor encoded by spo0A. In addition, the temperature-sensitive sporulation phenotype caused by the ftsA279 (spoIIN279) mutation was partly suppressed by the soj null mutation, indicating that FtsA might also affect the activity of Soj. Soj and Spo0J are known to be similar in sequence to a family of proteins involved in plasmid partitioning, including ParA and ParB of prophage P1, SopA and SopB of F, and IncC and KorB of RK2, spo0J was found to be required for normal chromosome partitioning as well as for sporulation. spo0J null mutants produced a significant proportion of anucleate cells during vegetative growth. The dual functions of Spo0J could provide a mechanism for regulating the initiation of sporulation in response to activity of the chromosome partition machinery.

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

Abstract: Genes coding for poly-beta-hydroxybutyrate were removed from Alcaligenes eutrophus H16 and cloned into Escherichia coli. Some of the clones produced PHB to 90% of the cell weight.

Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase.

Abstract: Melanin production is a major virulence factor for Cryptococcus neoformans, an organism causing life-threatening infections in an estimated 10% of AIDS patients. In order to characterize the events involved in melanin synthesis, an enzyme having diphenol oxidase activity was purified and its gene was cloned. The enzyme was purified as a glycosylated 75-kDa protein which migrated at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after deglycosylation by endoglycosidase F. Substrate specificity resembled that of a laccase in that it oxidized multiple diphenolic and diamino compounds. Dopamine was shown by mass spectroscopy to be oxidized to decarboxy dopachrome, an intermediate of melanin synthesis. The enzyme contained 4.1 +/- 0.1 mol of copper per mol. It resembled a laccase in its absorbance spectrum, containing a peak of 610 nm and the shoulder at 320 nm, corresponding to the absorbance of a type I and type III copper, respectively. The cloned gene of C. neoformans laccase (CNLAC1) contained a single open reading frame encoding a polypeptide 624 amino acids in length. The encoded polypeptide contained a presumptive leader sequence, on the basis of its relative hydrophobicity and by comparison of the sequence to that of the N-terminal sequence of the purified enzyme. CNLAC1 also contained 14 introns ranging from 52 to 340 bases long. Transcriptional activity of CNLAC1 was found to be derepressed in the absence of glucose and to correspond to an increase in enzymatic activity.

Phylogeny of mycoplasmalike organisms (Phytoplasmas): a basis for their classification.

Abstract: A global phylogenetic analysis using parsimony of 16S rRNA gene sequences from 46 mollicutes, 19 mycoplasmalike organisms (MLOs) (new trivial name, phytoplasmas), and several related bacteria placed the MLOs definitively among the members of the class Mollicutes and revealed that MLOs form a large discrete monophyletic clade, paraphyletic to the Acholeplasma species, within the Anaeroplasma clade. Within the MLO clade resolved in the global mollicutes phylogeny and a comprehensive MLO phylogeny derived by parsimony analyses of 16S rRNA gene sequences from 30 diverse MLOs representative of nearly all known distinct MLO groups, five major phylogenetic groups with a total of 11 distinct subclades (monophyletic groups or taxa) could be recognized. These MLO subclades (roman numerals) and designated type strains were as follows: i, Maryland aster yellows AY1; ii, apple proliferation AP-A; iii, peanut witches9-broom PnWB; iv, Canada peach X CX; v, rice yellow dwarf RYD; vi, pigeon pea witches9-broom PPWB; vii, palm lethal yellowing LY; viii, ash yellows AshY; ix, clover proliferation CP; x, elm yellows EY; and xi, loofah witches9-broom LfWB. The designations of subclades and their phylogenetic positions within the MLO clade were supported by a congruent phylogeny derived by parsimony analyses of ribosomal protein L22 gene sequences from most representative MLOs. On the basis of the phylogenies inferred in the present study, we propose that MLOs should be represented taxonomically at the minimal level of genus and that each phylogenetically distinct MLO subclade identified should represent at least a distinct species under this new genus. Images

A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Abstract: Seventeen fully sequenced and two partially sequenced extracytoplasmic proteins of purple, gram-negative bacteria constitute a homologous family termed the putative membrane fusion protein (MFP) family. Each such protein apparently functions in conjunction with a cytoplasmic membrane transporter of the ATP-binding cassette family, major facilitator superfamily, or heavy metal resistance/nodulation/cell division family to facilitate transport of proteins, peptides, drugs, or carbohydrates across the two membranes of the gram-negative bacterial cell envelope. Evidence suggests that at least some of these transport systems also function in conjunction with a distinct outer membrane protein. We report here that the phylogenies of these proteins correlate with the types of transport systems with which they function as well as with the natures of the substrates transported. Characterization of the MFPs with respect to secondary structure, average hydropathy, and average similarity provides circumstantial evidence as to how they may allow localized fusion of the two gram-negative bacterial cell membranes. The membrane fusion protein of simian virus 5 is shown to exhibit significant sequence similarity to representative bacterial MFPs.

The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons.

Abstract: The Escherichia coli flhD operon encodes two genes, flhD and flhC. Both gene products were overproduced and purified. The purified proteins formed a complex consisting of two FlhD and two FlhC molecules. Mobility shift assays showed that the FlhD/FlhC complex had a DNA-binding activity and bound to the upstream regions of fliA, flhB, and fliL operons (class II), which are under direct control of the flhD operon. DNase I footprinting analyses of FlhD/FlhC binding to the three class II promoter regions revealed protection of a 48-bp region of the fliA operon between positions -41 to -88, a 50-bp region of the flhB operon between positions -28 to -77, and a 48-bp region of the fliL operon between positions -29 to -76. In vitro transcription experiments demonstrated that the FlhD/FlhC complex is a transcriptional activator required for the transcription of the three class II operons examined in vitro.

Glycine betaine confers enhanced osmotolerance and cryotolerance on Listeria monocytogenes.

Abstract: Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.

Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster

Abstract: Escherichia coli K-12 has long been known not to produce an O antigen. We recently identified two independent mutations in different lineages of K-12 which had led to loss of O antigen synthesis (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994) and constructed a strain with all rfb (O antigen) genes intact which synthesized a variant of O antigen O16, giving cross-reaction with anti-O17 antibody. We determined the structure of this O antigen to be -->2)-beta-D-Galf-(1-->6)-alpha-D-Glcp- (1-->3)-alpha-L-Rhap-(1-->3)-alpha-D-GlcpNAc-(1-->, with an O-acetyl group on C-2 of the rhamnose and a side chain alpha-D-Glcp on C-6 of GlcNAc. O antigen synthesis is rfe dependent, and D-GlcpNAc is the first sugar of the biological repeat unit. We sequenced the rfb (O antigen) gene cluster and found 11 open reading frames. Four rhamnose pathway genes are identified by similarity to those of other strains, the rhamnose transferase gene is identified by assay of its product, and the identities of other genes are predicted with various degrees of confidence. We interpret earlier observations on interaction between the rfb region of Escherichia coli K-12 and those of E. coli O4 and E. coli Flexneri. All K-12 rfb genes were of low G+C content for E. coli. The rhamnose pathway genes were similar in sequence to those of (Shigella) Dysenteriae 1 and Flexneri, but the other genes showed distant or no similarity. We suggest that the K-12 gene cluster is a member of a family of rfb gene clusters, including those of Dysenteriae 1 and Flexneri, which evolved outside E. coli and was acquired by lateral gene transfer.

CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis.

Abstract: The dnaK and groESL operons of Bacillus subtilis are preceded by a potential sigma 43 promoter sequence (recognized by the vegetative sigma factor) and by an inverted repeat (IR) consisting of 9 bp separated by a 9-bp spacer. Since this IR has been found in many bacterial species, we suspected that it might be involved in heat shock regulation. In order to test this hypothesis, three different mutational alterations of three bases were introduced within the IR preceding the dnaK operon. These mutations were crossed into the chromosome of B. subtilis, and expression of the dnaK and of the unlinked groESL operons was studied. The dnaK operon exhibited increased expression at low temperature and a reduction in the stimulation after temperature upshift. Furthermore, these mutations reduced expression of the groESL operon at low temperature by 50% but did not interfere with stimulation after heat shock. These experiments show that the IR acts as a negative cis element of the dnaK operon. This conclusion was strengthened by the observation that the IR reduced expression of two different transcriptional fusions significantly after its insertion between the promoter and the reporter gene. Since this IR has been described in many bacterial species as preceding only genes of the dnaK and groESL operons, both encoding molecular chaperones (39 cases are documented so far), we designated this heat shock element CIRCE (controlling IR of chaperone expression). Furthermore, we suggest that this novel mechanism is more widespread among eubacteria than the regulation mechanism described for Escherichia coli and has a more ancient origin. Images

Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.

Abstract: The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE9s full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates. Images

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

Abstract: The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin. Images

Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes.

Abstract: The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors. In P. syringae pv. syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion. The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold. The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P. syringae pv. phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems. HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity. A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P. aeruginosa, as well as with the related alternate sigma factors was identified within region II. Key domains are partially conserved. Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold. Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually. The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes. The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes.

Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants.

Abstract: Pheromone-responsive conjugative plasmids are unique to the species Enterococcus faecalis. Many pheromone-responsive plasmids, including those frequently isolated from sites of infection, express a novel cytolysin that possesses both hemolytic and bacteriocin activities. Further, this cytolysin has been shown to be a toxin in several disease models. In the present study, nucleotide sequence determination, mutagenesis, and complementation analysis were used to determine the organization of the E. faecalis plasmid pAD1 cytolysin determinant. Four open reading frames are required for expression of the cytolysin precursor (cylLL, cylLS, cylM, and cylB). The inferred products of two of these open reading frames, CyILL and CyILS, constitute the cytolysin precursor and bear structural resemblance to posttranslationally modified bacteriocins termed lantibiotics. Similarities between the organization of the E. faecalis cytolysin determinant and expression units for lantibiotics exist, indicating that the E. faecalis cytolysin represents a new branch of this class and is the first known to possess toxin activity. Images

Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization.

Abstract: The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains. Images

YscN, the putative energizer of the Yersinia Yop secretion machinery.

Abstract: Pathogenic yersiniae secrete a set of 11 antihost proteins called Yops. Yop secretion appears as the archetype of the type III secretion pathway. Several components of this machinery are encoded by the virA (lcrA) and virC (lcrC) loci of the 70-kb pYV plasmid. In this paper, we describe yscN, another gene involved in this pathway. It is the first gene of the virB locus. It encodes a 47.8-kDa protein similar to the catalytic subunits of F0F1 and related ATPases, as well as to products of other genes presumed to be involved in a type III secretion pathway. YscN contains the two consensus nucleotide-binding motifs (boxes A and B) described by Walker et al. (J. E. Walker, M. Saraste, M. J. Runswick, and N. J. Gay, EMBO J. 1:945-951, 1982). We engineered a pYV mutant encoding a modified YscN protein lacking box A. This mutant, impaired in Yop secretion, can be complemented in trans by a cloned yscN gene. We conclude that YscN is a component of the Yop secretion machinery using ATP. We hypothesize that it is either the energizer of this machinery or a part of it.

Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density.

Abstract: We have identified a gene that acts in trans to activate the expression of the phenazine biosynthetic genes in the biological control organism Pseudomonas aureofaciens 30-84 This gene, phzR (phenazine regulator), is located upstream of and divergently transcribed from the phenazine biosynthetic genes Thus, the phenazine biosynthetic locus consists of at least two divergently transcribed operons A functional phzR gene is required for phenazine production The nucleotide sequence of phzR revealed an open reading frame of 723 nucleotides encoding a protein of ca 27 kDa The predicted amino acid sequence of PhzR has homology with other bacterial positive transcriptional activators, including LasR of Pseudomonas aeruginosa, LuxR of Vibrio fischerii, and TraR of Agrobacterium tumefaciens The addition of cell-free supernatants from late-exponential-phase cultures of strain 30-84 resulted in expression of a genomic phzB:lacZ reporter strain at a lower cell density than normal, indicating the possible presence of an autoinducer These results indicate that PhzR is a member of a two-component sensor-regulator family with known or predicted carboxy-terminal DNA-binding domains which regulates gene expression in response to environmental and cell density signals

Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol.

Abstract: The metabolism of Clostridium acetobutylicum was manipulated, at neutral pH and in chemostat culture, by changing the overall degree of reduction of the substrate, using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids, and the intracellular enzymatic pattern indicated the absence of butyraldehyde dehydrogenase activity and very low levels of coenzyme A-transferase, butanol, and ethanol dehydrogenase activities. In contrast, cultures grown on mixtures of glucose and glycerol produced mainly alcohols and low levels of hydrogen. The low production of hydrogen was not associated with a change in the hydrogenase level but was correlated with the induction of a ferredoxin-NAD reductase and a decreased level of NADH-ferredoxin reductase. The production of alcohols was related to the induction of a NAD-dependent butyraldehyde dehydrogenase and to higher expression of NAD-dependent ethanol and butanol dehydrogenases. The coenzyme A-transferase was poorly expressed, and thus no acetone was produced. These changes in the enzymatic pattern, obtained with cultures grown on a mixture of glucose and glycerol, were associated with a 7-fold increase of the intracellular level of NADH and a 2.5-fold increase of the level of ATP.

prrA, a putative response regulator involved in oxygen regulation of photosynthesis gene expression in Rhodobacter sphaeroides.

Abstract: A new locus, prrA, involved in the regulation of photosynthesis gene expression in response to oxygen, has been identified in Rhodobacter sphaeroides. Inactivation of prrA results in the absence of photosynthetic spectral complexes. The prrA gene product has strong homology to response regulators associated with signal transduction in other prokaryotes. When prrA is present in multiple copies, cells produce light-harvesting complexes under aerobic growth conditions, suggesting that prrA affects photosynthesis gene expression positively in response to oxygen deprivation. Analysis of the expression of puc::lacZ fusions in wild-type and PrrA- cells revealed a substantial decrease in LacZ expression in the absence of prrA under all conditions of growth, especially when cells were grown anaerobically in the dark in the presence of dimethyl sulfoxide. Northern (RNA) and slot blot hybridizations confirmed the beta-galactoside results for puc and revealed additional positive regulation of puf, puhA, and cycA by PrrA. The effect of truncated PrrA on photosynthesis gene expression in the presence of low oxygen levels can be explained by assuming that PrrA may be effective as a multimer. PrrA was found to act on the downstream regulatory sequences (J. K. Lee and S. Kaplan, J. Bacteriol. 174:1146-1157, 1992) of the puc operon regulatory region. Finally, two spontaneous prrA mutations that abolish prrA function by changing amino acids in the amino-terminal domain of the protein were isolated.

Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes.

Abstract: The Agrobacterium tumefaciens virB gene products are proposed to assemble into a transport system capable of exporting complexes of DNA and protein across the bacterial envelope en route to plant cells. Nonpolar null mutations were constructed in each of the 11 virB genes of the A. tumefaciens pTiA6NC plasmid. In tumorigenicity assays, delta virB1 mutants exhibited severely attenuated virulence and delta virB2 through delta virB11 mutants exhibited avirulence. NdeI restriction sites introduced at the predicted translational start sites of the virB genes were used to subclone each of the virB genes downstream of the lacZ or virB promoter on broad-host-range plasmids. virB gene expression plasmids were used to define promoter and general sequence requirements for genetic complementation of the deletion mutations. Whereas virB1 and virB2 complemented delta virB1 and delta virB2, respectively, only when expressed in trans from the virB promoter, virB3 through virB11 complemented the corresponding deletion mutations when expressed in trans from either the lacZ or virB promoter. Several virB genes required additional upstream or downstream sequences for complementation: (i) virB2 complemented the delta virB2 mutation only when the complementing plasmid coexpressed virB1 and virB2, (ii) virB6 and virB9 complemented the delta virB6 and delta virB9 mutations only when the complementing plasmids carried at most 55 and 230 bp of sequences residing 5' of these genes, respectively, and (iii) virB7 and virB8 complemented the delta virB7 and delta virB8 mutations only when the complementing plasmid coexpressed virB7 and virB8. These studies established that virB1 is an accessory virulence determinant and virB2 through virB11 are absolutely essential for the A. tumefaciens infection process.

Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors.

Abstract: The conversion of Pseudomonas aeruginosa into the mucoid, exopolysaccharide alginate-overproducing form is considered to be a major pathogenic determinant expressed by this organism during chronic respiratory infections in cystic fibrosis (CF) (5, 28, 32). Despite the fact that CF has a relatively recent history as a medically recognized syndrome (since 1938), the problem of conversion to mucoidy in P. aeruginosa has become an instant classic of microbial pathogenesis (for recent reviews, see references 28, 54, and 61). Most P. aeruginosa strains have the genetic capacity to synthesize alginate, but mucoid mutants that overproduce this exopolysaccharide are rarely noticed among environmental isolates. In contrast, P. aeruginosa strains from CF are frequently mucoid (28, 32). Several mutations causing conversion to mucoidy (see Fig. 1) and genes that participate in these processes have been recently defined (see Table 1); the mutations are superimposed on a system consisting of a putative alternative sigma factor, which most likely plays a more general role in bacterial stress response, and its accessory negative regulators (23, 30, 51-53, 61).