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Transcription from promoter Pu of the upper catabolic operon of the Pseudomonas putida TOL plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the TOL‐encoded regulator XylR protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(NtrA)‐containing form of RNA polymerase. This regulatory circuit was faithfully reproduced in Escherichia coli in single copy gene dosage by integrating the corresponding controlling determinants into the chromosomes of several K12 derivatives by means of specialized transposons. In vivo monitoring of the activity of a Pu‐lacZ fusion in E. coli strains with different genetic backgrounds demonstrated that integration host factor (IHF) is involved in Pu regulation and that hyperproduction of the XylR protein leads to a decrease of Pu activity in a manner in which deletion of the putative DNA‐binding domain of the XylR does not impair its inhibitory effect when hyperproduced. One discrete IHF binding site and two potential XylR sites (consensus sequence 5′‐TTGANCAAATC‐3′), bracketted by short stretches of DNase I‐hypersensitive bonds, were detected upstream of the transcription initiation site. A model accounting for the features found is proposed which includes the IHF‐promoted looping of upstream XylR‐DNA complexes so that they contact the sigma 54(NtrA)‐RNA polymerase bound at ‐12/‐24 positions.

Cells of Escherichia coli K–12 containing a cloned fragment of Pseudomonas putida TOL plasmid pWW0 produce the dye indigo. Analysis of the cloned fragment and Tn1000 transposon insertion mutagenesis has identified the xylA gene as being responsible for this phenotype. The xylA gene specifies xylene oxidase, a relaxed specificity enzyme that hydroxylates or monooxygenates toluene and xylenes and their corresponding alcohols. Indole, which is formed from tryptophan by tryptophanase in E. coli, was shown to be a precursor in the reaction sequence leading to indigo formation. These results suggest a novel route for bacterial production of indigo via hydroxylation of indole.

Transcription from promoter Pu of the upper catabolic operon of the Pseudomonas putida TOL plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the TOL‐encoded regulator XylR protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(NtrA)‐containing form of RNA polymerase. This regulatory circuit was faithfully reproduced in Escherichia coli in single copy gene dosage by integrating the corresponding controlling determinants into the chromosomes of several K12 derivatives by means of specialized transposons. In vivo monitoring of the activity of a Pu‐lacZ fusion in E. coli strains with different genetic backgrounds demonstrated that integration host factor (IHF) is involved in Pu regulation and that hyperproduction of the XylR protein leads to a decrease of Pu activity in a manner in which deletion of the putative DNA‐binding domain of the XylR does not impair its inhibitory effect when hyperproduced. One discrete IHF binding site and two potential XylR sites (consensus sequence 5′‐TTGANCAAATC‐3′), bracketted by short stretches of DNase I‐hypersensitive bonds, were detected upstream of the transcription initiation site. A model accounting for the features found is proposed which includes the IHF‐promoted looping of upstream XylR‐DNA complexes so that they contact the sigma 54(NtrA)‐RNA polymerase bound at ‐12/‐24 positions.

The beta-antigen of the c protein complex of group B streptococci contains two immunoglobulin A (IgA)-binding domains called A and B. A 73-amino-acid segment in domain A is responsible for most of the IgA-binding activity. To identify the IgA binding motif, the 73-amino-acid domain was divided into 60 14-amino-acid overlapping peptides spot synthesized onto a cellulose membrane. A 20-residue putative antigenic epitope was identified and expressed as a fusion protein. The fusion protein was purified by fast protein liquid chromatography and used to raise rabbit antiserum. By use of a membrane with spot-synthesized peptide amino acids of decreasing length (from 14 to 6 amino acids), the major antigenic epitope recognized by the anti-fusion protein antibodies was mapped to motif MLKKIE. Anti-fusion protein antibodies inhibited the binding of IgA to group B streptococci. This inhibition could be blocked by the peptide containing the motif MLKKIE. These results indicate that the motif MLKKIE is located in the IgA-binding site. The IgA-binding domain of beta-antigen from three group B streptococcal strains reacted with the anti-fusion protein antibodies, and their coding sequences gave positive signals in Southern hybridization. The sequences of beta-antigen from these strains were amplified by PCR, and sequence analysis showed them to be identical. The results indicate that the motif MLKKIE is required for IgA binding and is present in different group B streptococcal strains.

The isolation of a series of plasmid mutant derivatives that overproduce the traT outer membrane protein, TraTp, is described. Some of the mutants directed the synthesis of 10-fold more TraTp (200,000 copies/cell) than did the parental plasmid (20,000 copies/cell). The proteins specified by all mutant plasmids except one were correctly inserted into the outer membrane and exposed on the cell surface. The TraTp that was not correctly inserted did not mediate the expected levels of surface exclusion and serum resistance, suggesting that surface localization is a requirement of TraTp function. The overproduction of TraTp was deleterious to bacterial growth, particularly that of minicell mutants of E. coli K-12.

Catechol 2,3-dioxygenase (XylE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. Purified XylE is oxygen-sensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE. In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyperexpressible xylT gene and characterized it as a novel [2Fe-2S] ferredoxin. It is the first example of a soluble ferredoxin with a net positive charge at neutral pH. The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin. The midpoint redox potential was determined to be -373 +/- 6 mV, at pH 8. 5. XylT was unusually unstable for a [2Fe-2S] ferredoxin, with half-lives of 69 min at 25 degrees C in air and 70 min at 37 degrees C in argon. With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro. Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR. Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme. It is the first instance of a ferredoxin-mediated reactivation of an enzyme. The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT.

The O-antigen polysaccharide of the lipopolysaccharide of Shigella dysenteriae serotype 1 is encoded by determinants located on a 9 kb plasmid (rfp) and on the chromosome near the his locus (rfb). Molecular genetic and biochemical studies of the rfp determinant reported here show that the rfp region contains two genes, rfpA and rfpB, lying in an operon. rfpB was demonstrated to encode a membrane bound galactosyl transferase. The low G+C content of rfp DNA suggests that it did not originate in Shigella. Copyright 1994, 1999 Academic Press

Sfb protein, a fibronectin binding adhesin of Streptococcus pyogenes (Lancefield group A streptococcus), mediates streptococcal adherence to human epithelial cells via its fibronectin binding domain coded by a repetitive gene region named fnbr. In the present study, Southern blot analysis using the fnbr gene region as a probe to screen genomic DNA from 51 epidemiologically unrelated clinical isolates of S. pyogenes revealed that 70% carried a sequence homologous to the fnbr probe. Among ten other streptococcal strains belonging to serological groups B, C, and G, DNA from only two human S. equisimilis (group C) strains reacted with the probe. Further analysis by PCR-mediated amplification of the binding repeat coding sequences revealed that repeats of different S. pyogenes isolates were identical in size but varied in number, ranging from one to five. Most of the isolates were shown to carry multiple repeats. Presence of the probe-positive sequence correlated strongly with streptococcal binding to purified fibronectin and adherence to HEp2 human epithelial cells; of the 36 probe-positive isolates, 95% bound fibronectin and 89% adhered strongly to epithelial cells, whereas among the 15 probe-negative isolates only 27% had binding actvities for fibronectin and 27% showed strong adherence to HEp2 cells. Antibodies raised against the fibronectin binding domain of Sfb protein recognized streptococcal fibronectin binding surface proteins in most of the clinical isolates but did not react with heart or skeletal muscle myosin in an enzyme immunoassay, as is the case with antibodies directed to M protein, another major surface protein of group A streptococci. The results of the present study suggest that Sfb protein could be a potential candidate for a streptococcal vaccine. Copyright 1994, 1999 Academic Press

A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.

A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).

Mutant derivatives of the TOL plasmid pWW0-161, containing Tn5 insertions in the xylS and xylR regulatory genes of the catabolic pathway, have been identified and characterized. The two genes are located together on a 1.5- to 3.0-kilobase segment of TOL, just downstream of genes of the enzymes of the meta-cleavage pathway. As predicted by a current model for regulation of the TOL catabolic pathway, benzyl alcohol dehydrogenase, a representative enzyme of the upper (hydrocarbon leads to carboxylic acid) pathway, was induced by m-methylbenzyl alcohol in xylS mutant bacteria but not in a xylR mutant, whereas catechol 2,3-oxygenase, a representative enzyme of the lower (meta-cleavage) pathway, was induced by m-toluate in a xylR mutant but not in the xylS mutants. Unexpectedly, however, catechol 2,3-oxygenase was not induced by m-methylbenzyl alcohol in xylS mutants but was induced by benzyl alcohol and benzoate. These results indicate that expression of the TOL plasmid-encoded catabolic pathway is regulated by at least three control elements, two of which (the products of the xylS and xylR genes) interact in the induction of the lower pathway by methylated hydrocarbons and alcohols and one of which responds only to nonmethylated substrates.

Nine high copy number mutations of plasmid R6-5, representing five phenotypically distinct groups, have been identified by DNA sequencing. In each mutant plasmid examined, a single nucleotide change was found. The effects of the mutations on possible gene products, and DNA-RNA secondary structure, were analyzed and compared with the observed phenotypes. The results of this study exclude the possibility that the primary plasmid replication control element, the product of the copA gene, is a polypeptide, and they are consistent with a model of plasmid replication control by the copA product which has the following features: (i) RNA-I, a short untranslated RNA molecule, is the product of the copA gene and regulates the frequency of initiation of plasmid replication, (ii) the hexanucleotide single-strand loop of the major hairpin of RNA-I is its active site, (iii) this active site functions by base pair interactions with its "target," its DNA template strand, or its complementary sequence on RNA-II, a transcript of opposite polarity that is the message of the repA gene, and (iv) the sequence and size of the loop, and the stability of the stem of the hairpin, are all critical factors that govern the functioning of RNA-I.

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.

A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K. In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication. This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria. Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability.

The spread of Shigella flexneri in a monolayer of infected Henle and HeLa cells was studied by using immunofluorescence and electron microscopy. Infected cells produced numerous bacterium-containing membranous protrusions up to 18 microns in length that penetrated adjacent cells and were subsequently phagocytosed. Fluorescence staining of actin and vinculin in infected cells with phalloidin and monoclonal antibody to vinculin, respectively, demonstrated that the protrusions containing the bacteria consisted of these cytoskeletal proteins. Actin accumulated predominantly at the poles of bacteria distal to the tip of protrusions and appeared as trails extending back towards the host cell cytoplasm. Vinculin, however, was distributed uniformly around the bacteria and throughout the protrusion. A profound rearrangement of vinculin occurred in Henle and HeLa cells following infection with shigellae: whereas in uninfected cells it was distributed mainly around the cell periphery, in infected cells it concentrated mainly around clusters of bacteria in the cytoplasm. This suggests a possible involvement of the vinculin cytoskeletal protein in the intercellular spread of shigellae during an infection.

Serotype-specific fimbriae of Bordetella pertussis are considered potential components of new-generation vaccines against whooping cough. Attempts to characterize fimbriae, and indeed other virulence determinants, produced by B. pertussis have been frustrated on one hand by low yields from B. pertussis itself and on the other by an inability to produce native recombinant products in Escherichia coli. In order to try to circumvent this problem, we have examined the expression of B. pertussis serotype 2 fimbriae in Bordetella parapertussis and Bordetella bronchiseptica from native as well as E. coli expression signals. These studies revealed that the fimbrial gene product was expressed from the original B. pertussis promoter and Shine-Dalgarno sequence in both B. parapertussis and B. bronchiseptica. The transcriptional start site of the gene was located 146 nucleotides upstream of its ATG start codon. A recombinant fimbrial subunit gene containing PLAC and the atpE translation initiation region of E. coli was also expressed in B. bronchiseptica. In all cases in which gene expression was detected the gene product was expressed as serotype 2-specific fimbriae as determined by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopic investigation of the bacterial cell surface.

Chloroaromatics, a major class of industrial pollutants, may be oxidatively metabolized to chlorocatechols by soil and water microorganisms that have evolved catabolic activities toward these xenobiotics. We show here that 4-chlorocatechol can be further transformed by enzymes of the ubiquitous 3-oxoadipate pathway. However, whereas chloromuconate cycloisomerases catalyze the dechlorination of 3-chloro-cis,cis-muconate to form cis-dienelactone, muconate cycloisomerases catalyze a novel reaction, i.e. the dechlorination and concomitant decarboxylation to form 4-methylenebut-2-en-4-olide (protoanemonin), an ordinarily plant-derived antibiotic that is toxic to microorganisms.

The replication control system of plasmid R6-5 has been investigated by characterization of high-copy-number mutant miniplasmids, development of an in vivo assay for the site of action or "target" of the replication control elements, and sequence analysis of the replication control regions of the wild-type plasmid and two copy-number mutant derivatives. These and other experiments have shown that three plasmid determinants--copA/incA, copB, and copT--are involved in DNA replication control. The products of the copB and copA/incA genes, a 9500-dalton basic polypeptide and either a 7200-dalton basic polypeptide or a short untranslated RNA molecule, respectively, are negative-acting elements that interact with the third element, their target, the copT DNA sequence, or its product to regulate the frequency of initiation of plasmid replication. The location of copT within the copA/incA gene and 1600 base pairs upstream from the origin of replication indicates that regulation is effected at a preinitiation stage of replication, such as the production of a primer or other initiation factor.

A key enzyme in the degradation pathways of dibenzo-p-dioxin and dibenzofuran, namely, 2,2',3-trihydroxybiphenyl dioxygenase, which is responsible for meta cleavage of the first aromatic ring, has been genetically and biochemically analyzed. The dbfB gene of this enzyme has been cloned from a cosmid library of the dibenzo-p-dioxin- and dibenzofuran-degrading bacterium Sphingomonas sp. strain RW1 (R. M. Wittich, H. Wilkes, V. Sinnwell, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:1005-1010, 1992) and sequenced. The amino acid sequence of this enzyme is typical of those of extradiol dioxygenases. This enzyme, which is extremely oxygen labile, was purified anaerobically to apparent homogeneity from an Escherichia coli strain that had been engineered to hyperexpress dbfB. Unlike most extradiol dioxygenases, which have an oligomeric quaternary structure, the 2,2',3-trihydroxybiphenyl dioxygenase is a monomeric protein. Kinetic measurements with the purified enzyme produced similar Km values for 2,2',3-trihydroxybiphenyl and 2,3-dihydroxybiphenyl, and both of these compounds exhibited strong substrate inhibition. 2,2',3-Trihydroxydiphenyl ether, catechol, 3-methylcatechol, and 4-methylcatechol were oxidized less efficiently and 3,4-dihydroxybiphenyl was oxidized considerably less efficiently.

Transcriptional lacZ fusions to the Pu and Pm promoters of the TOL (toluene degradation) plasmid inserted in monocopy in the chromosome of Pseudomonas putida showed a very different responsiveness to their respective aromatic effectors regarding growth phase. While a substantial XylS-dependent activation of Pm-lacZ was detected nearly instantly after m-toluate addition, XylR- and xylene-mediated induction of the sigma 54 promoter Pu became significant only after cells slowed down exponential growth and entered stationary phase. When Pu and Pm were fused to lacZ-tet reporters (i.e., promoterless lacZ genes coupled to a tet gene which confers resistance to tetracycline when cotranscribed with the leading gene) instead of lacZ alone, the resulting colonies displayed a distinct phenotype consisting of hyperfluorescence on agar plates after being sprayed with 4-methylumbelliferyl-beta-D-galactoside, simultaneously with being either sensitive (Pu) or resistant (Pm) to tetracycline. To examine whether the same phenotype could be scored in strains carrying transcriptional fusions of the lacZ-tet cassettes to other genes or promoters whose expression is silenced during growth and activated in stationary phase, we constructed mini-Tn5 lacZ-tet transposons for random genetic probing of promoters preferentially active at advanced stages of growth. Chromosomal insertions of this mobile element were selected by means of the constitutive resistance to kanamycin which is also specified by the transposon. A number of kanamycin-resistant colonies which are hyperfluorescent with 4-methylumbelliferyl-beta-D-galactosidase but sensitive to tetracycline and which reached full induction only at postexponential growth stages were obtained.

A red-pigmented coryneform bacterium, identified as Rhodococcus rhodochrous strain 116, that grew on 2-ethoxyphenol and 4-methoxybenzoate as sole carbon and energy sources was isolated. Phylogenetic analysis based on the 16S rDNA sequences indicates that the strain clusters more closely to other rhodococci than to other gram-positive organisms with a high G + C content. Each of the abovementioned growth substrates was shown to induce a distinct cytochrome P-450: cytochrome P-450RR1 was induced by 2-ethoxyphenol, and cytochrome P-450RR2 was induced by 4-methoxybenzoate. A type I difference spectrum typical of substrate binding was induced in cytochrome P-450RR1 by both 2-ethoxyphenol (KS = 4.2 +/- 0.3 microM) and 2-methoxyphenol (KS = 2.0 +/- 0.1 microM), but not 4-methoxybenzoate or 4-ethoxybenzoate. Similarly, a type I difference spectrum was induced in cytochrome P-450RR2 by both 4-methoxybenzoate (KS = 2.1 +/- 0.1 microM) and 4-ethoxybenzoate (KS = 1.6 +/- 0.1 microM), but not 2-methoxyphenol or 2-ethoxyphenol. A purified polyclonal antiserum prepared against cytochrome P-450RR1 did not cross-react with cytochrome P-450RR2, indicating that the proteins are immunologically distinct. The cytochromes appear to catalyze the O-dealkylation of their respective substrates. The respective products of the O-dealkylation are further metabolized via ortho cleavage enzymes, whose expression is also regulated by the respective aromatic ethers.

Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5. The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli. The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis. This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon. The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated. Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS.

The TraT protein specified by IncF group plasmids mediates surface exclusion and bacterial resistance to the lethal activities of serum. In this study, an anti-TraT protein monoclonal antibody was generated which failed to react with TraT+ bacteria but which efficiently detected solubilized TraT protein in Western blots and in an enzyme-linked immunosorbent assay. Use of this antibody to screen clinical and nonclinical isolates of Escherichia coli for the production of TraT protein revealed its presence in a modest proportion (38%) of normal fecal strains, a significantly higher proportion of clinical strains (51 to 73%), and an even higher proportion (78 to 88%) of clinical strains concomitantly producing the K1 capsule, an important virulence factor of E. coli.

The presence of the outer membrane protein TraT, encoded by plasmid R6-5, reduces the sensitivity of Escherichia coli cells to phagocytosis by macrophages. This effect is independent of the bacterial capsule and is more evident in the presence of adsorbed normal human serum. The property of inhibiting phagocytosis is specifically abolished by anti-TraT protein antiserum and anti-TraT immunoglobulin G but not by Fab fragments. These results indicate that the TraT protein is a passive inhibitor of phagocytosis. Inhibition of phagocytosis is produced because the TraT protein antagonizes opsonization by complement, such that C3 deposition is reduced and altered in distribution.

Culture filtrates of two Aeromonas hydrophila strains which were isolated from patients with diarrhea and assumed to be causative agents of the infections were shown to contain enterotoxic, cytotoxic, and hemolytic activities. Modest heat treatment of the filtrates inactivated the cytotoxic and cytolytic activities, but not the enterotoxic activity. The construction of cosmid gene banks in Escherichia coli of DNA from both A. hydrophila strains demonstrated that the determinants of the three activities are located on three different segments of the A. hydrophila chromosome. Both heated culture filtrates of A. hydrophila and nonheated filtrates of an E. coli clone containing the A. hydrophila enterotoxin gene provoked fluid accumulation in the rabbit ileal loop and suckling mouse models and caused elongation of Chinese hamster ovary cells. Differences in the responses of the models to the A. hydrophila enterotoxin and to the heat-labile and heat-stabile toxins of E. coli indicated that the former is distinct from the latter two types of toxin. These results constitute conclusive evidence for the production by A. hydrophila of a cytotonic enterotoxin that is distinct from the A. hydrophila cytotoxin and hemolysin and known E. coli enterotoxins.

A 6-megadalton plasmid, pHW400, of Shigella dysenteriae 1 strain W30864 was previously found to specify one or more functions for O-antigen production and bacterial virulence (H. Watanabe and K. N. Timmis, Infect. Immun. 43:391-396, 1984). The region of pHW401, a Tn801-tagged derivative of pHW400, responsible for O-antigen production has been localized by gene cloning and Tn5 transposon mutagenesis. Analysis of lipopolysaccharide isolated from S. dysenteriae 1 bacteria carrying mutant plasmids revealed that the determinant for O-antigen synthesis, designated rfp, codes for a function involved in the formation of the O-polysaccharide side chain structure of lipopolysaccharide. Analysis of radioactively labeled proteins synthesized in minicells of Escherichia coli carrying mutant plasmids identified the product of the rfp gene as a 41,000-dalton protein. Southern hybridization with a DNA fragment carrying the rfp gene demonstrated that this determinant is present on 6-megadalton plasmids in other isolates of S. dysenteriae 1 but is not present at all in a variety of other Shigella, E. coli, and Salmonella typhimurium strains that were tested.

In this study, the interaction of murine dendritic cells with Listeria monocytogenes was investigated. Dendritic cells are efficient antigen-presenting cells, play a key role in the immune response, and are capable of migrating over substantial distances between sites of infection and lymphoid tissues. L. monocytogenes EGD invaded dendritic cells, escaped from phagosomes into the cytoplasm, and there directed actin nucleation, polymerization, and polarization in a typical fashion, thereby achieving intracellular movement and cell-to-cell spread. The internalization process appears to be independent of the inl locus. Interestingly, an intact microtubular function was essential for efficient uptake, whereas in a previous report, microtubule disruption did not affect bacterial spread in Caco-2 cells. The results obtained also suggest that L. monocytogenes binds to glycosylated receptors of dendritic cells. Uptake of Listeria cells was mediated by a protein kinase-dependent transducing phosphorylation signal that induces the actin polymerization-dependent phagocytic process. To achieve efficient uptake, de novo protein synthesis of eukaryotic and prokaryotic cells is also required. Despite the killing of dendritic cells, wild-type bacteria were found to persist in small numbers in some cells for at least 24 h. When different isogenic mutants of the EGD strain were analyzed for their capability to interact with dendritic cells, it was observed that some virulence-attenuated mutants (i.e., prfA and delta hly) persisted in large numbers for even longer times. Invasion of dendritic cells by L. monocytogenes, which in turn could result in either cell death or persistent infection, might have an important role in the pathogenesis of listeriosis, leading to impaired immune responses with inefficient bacterial clearance and/or promoting bacterial spread.

Mice were orally vaccinated with liposomes coated with filamentous hemagglutinin (FHA) and detoxified pertussis toxin (PT) of Bordetella pertussis. FHA- and PT-specific immunoglobulin G (IgG) was detected in serum, and both IgG and IgA were detected in lung washes following the immunization. Antibody responses in mice immunized with liposomes coated with FHA and PT were significantly higher than those in mice immunized with free FHA and PT, which demonstrated the adjuvanticity of the liposome carrier. The results indicate the potential usefulness of this approach for eliciting immune responses against FHA and PT (and perhaps other pertussis antigens) in humans and its possible utility in large-scale vaccination to protect against both B. pertussis infection and disease.

The filamentous hemagglutinin (FHA) of Bordetella pertussis was expressed in the attenuated aroA mutant of Salmonella typhimurium, SL3261, and in a strain of Escherichia coli harboring Shigella flexneri plasmid pWR110, which encodes bacterial invasiveness for epithelial cells. Expression of FHA in these strains did not interfere with their ability to invade Henle cells. Immunoglobulins A and G specific for FHA were detected in lung washes of mice following oral immunization with the live recombinant organisms; antibody levels were significantly higher than those in mice immunized with killed bacteria administered orally or intraperitoneally. Live oral vaccines carrying protective antigens of B. pertussis may be an important alternative to new-generation component vaccines against whooping cough.

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations.

Nonfused (i.e., nonhybrid) filamentous hemagglutinin (FHA) of Bordetella pertussis was efficiently expressed in Escherichia coli K-12 and Salmonella typhimurium aroA at levels higher than those found in wild-type B. pertussis when the upstream signals of the gene were replaced and the translation initiation region was engineered to optimize translational efficiency. Inclusion of part of the C-terminal FHA open reading frame, whose translation product does not appear to be part of the major secreted species of FHA, was shown to be important in achieving protein expression in both E. coli and S. typhimurium aroA; removal of the downstream gene sequence abolished recombinant FHA production. The levels of expression observed varied widely according to the construct and host bacterium used.

Adherence of group A, B, and C streptococci to fibrin thrombi was studied by using a novel fluorochrome microassay carried out in microdilution plates in which fibrin thrombi had been prepared by clotting citrated human, cattle, or horse plasma. Substantial adherence was observed with various strains of group A and C streptococci, whereas little was observed with group B streptococci. Adherence of all group A and C streptococcal strains decreased by up to 40% when fibronectin was depleted from the plasmas used for preparing fibrin thrombi, and fibronectin repletion increased adherence of streptococci in a dose-dependent manner. Addition of the 210-kilodalton C-terminal fragment of fibronectin to fibronectin-depleted plasma restored the adherence of group C but not group A streptococci, whereas addition of the 29-kilodalton N-terminal fragment was without any effect for all tested streptococcal strains. Prior incubation of group A and C streptococcal strains with fibronectin markedly increased their adherence, but treatment with proteases abolished their ability to adhere to fibrin thrombi. Adherence of group B streptococci was not affected by either fibronectin depletion or proteolytic digestion. These results indicate that both fibronectin incorporated into the fibrin matrix of thrombi and soluble fibronectin can mediate adherence of group A and C streptococci to fibrin thrombi and that binding sites for fibronectin located on the bacterial surface mediate this adherence.

Genes coding for production of the K1 polysaccharide capsule of Escherichia coli have been cloned. Complementation, insertion, and deletion analyses were used to localize the K1 genes and demonstrated that a minimum of 9 kilobases of DNA split into at least two gene blocks is involved in synthesis and assembly of the capsule. One of the gene blocks is responsible for biosynthesis of the polysaccharide, and the other is responsible for extracellular appearance of capsular material. Using cloned K1 genes as probes in Southern blot experiments, we detected homology to DNA from strains of E. coli capsular types K92, K7, and K100. In contrast, no homology was apparent between K1 genes and DNA from meningococcus group B, although the K1 and group B capsules are chemically and immunologically identical.

EcoRI endonuclease-generated DNA fragments carrying replication regions of the F'lac and R6-5 plasmids have been cloned and isolated, using as a selection vehicle a nonreplicating ampicillin-resistance DNA fragment derived from a Staphylococcus aureus plasmid. Heteroduplex analysis of the constructed plasmid chimeras and the parent replicons has localized the cloned R6-5 replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical proximity between the plasmid replication functions and the locus governing plasmid incompatibility has been shown for both parent replicons. The cloning method reported appears to be generally applicable for the identification and isolation of replication regions of a variety of complex genomes.

The genetic organization of the Pseudomonas putida plasmid pWWO-161, which encodes enzymes for the degradation of toluene and related aromatic hydrocarbons, has been investigated by transposition mutagenesis and gene cloning. Catabolic genes were localized to two clusters, one for upper pathway (hydrocarbon leads to carboxylic acid) enzymes and the other for lower pathway (carboxylic acid leads to tricarboxylic acid cycle) enzymes, that are separated by a 14-kilobase DNA segment. The physical organization of the catabolic genes thus reflects their functional organization into two regulatory blocks. The pWWO-161 DNA fragments Sst I fragment C and fragment D were cloned in a broad host range vector to produce plasmid pKT530. This hybrid encodes toluate oxygenase and all meta cleavage pathway enzymes, and it enables P. putida mt-2 and Escherichia coli K-12 cells to grow on m-toluate as sole carbon source. The pKT530 plasmid also carries xylS (a gene whose product has been postulated to regulate expression of the lower pathway genes) and the control sequences of the pathway that interact with this product, because catechol 2,3-oxygenase synthesis is specifically induced by m-toluate in both P. putida and E. coli. Evidence is presented that suggests the promoter operator of the meta pathway gene functions less effectively with the RNA polymerase or xylS product of E. coli than with the enzyme or product of P. putida.

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon Tn501 into the Shigella chromosome through Tn501-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage P1-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stx- mutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a Tn10 derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stx- mutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycin-resistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx- mutants of S. dysenteriae 1 exhibited full virulence in the Serény test.

Total-cell DNA isolated from a highly virulent serotype Ic strain of a group B streptococcus was used to construct a gene bank with bacteriophage lambda L47.1 in Escherichia coli K-12. Recombinant phage plaques in the bank were immunoblotted by using anti-alpha- and anti-beta-specific antibodies directed towards the Ibc proteins purified from the streptococcal cell surface, and hybrid phages expressing the alpha protein (lambda alpha +) and the beta protein (lambda beta +) were identified. DNA inserts in these phages were subcloned into E. coli high-copy-number plasmid vectors to produce stable alpha + (pPHC10) and beta + (pPHC8 and pPHC33) recombinant plasmids, and restriction maps of the cloned streptococcal sequences were constructed. Antibodies against the two Streptococcus-derived proteins reacted with high-molecular-weight polypeptides made in E. coli cells carrying the corresponding hybrid plasmids and with several degradation peptides from them. A 190-kilodalton alpha protein, previously undetected, was identified; this species may be the native alpha protein or a precursor of it. In addition, mutagenesis of the cloned sequences was carried out by using the omega fragment to determine the direction of transcription. In E. coli, the beta protein, but not the alpha protein, bound human immunoglobulin A (IgA) in Western blots, and neither protein bound IgG or IgM.

A gene bank of group A Streptococcus strain Manfredo (M protein serotype 5) was constructed with a bacteriophage lambda vector-Escherichia coli K-12 host system and screened by immunoblotting hybrid phage plaques with antisera raised to purified pep M5 (serotype 5 M protein fragment released from the streptococcal cell surface by pepsin). Hybrid phage expressing M5 antigen (lambda M5) were detected in the gene bank at an unexpectedly high frequency. The cloned streptococcal DNA sequences from one lambda M5 phage were subcloned into an E. coli plasmid vector. The M5 gene (smp5) was mapped, and its transcriptional orientation was determined by isolating and characterizing deletion and transposon insertion mutants of the M5+ hybrid plasmid pMK207. This analysis indicated that the intact smp5 gene had been cloned. Anti-pep M5 sera reacted with five pMK207-encoded polypeptides having relative molecular sizes of 64,000, 56,000, 55,500, 52,500, and 50,000. All of these polypeptides were encoded by the same DNA sequences, and all reacted with antisera raised to a synthetic peptide corresponding to the amino-terminal end of pep M5, suggesting that proteolytic cleavage at the carboxy-terminal end of the smp5 gene product generates at least some of the lower relative molecular size forms. Southern blotting experiments with smp5 gene sequences as probes identified multiple copies of DNA sequences sharing homology with the smp5 gene in the type 5 group A streptococcal genome.

The immunochemical properties of type 5 M protein antigens that were expressed in Escherichia coli K-12 by recombinant lambda bacteriophages isolated from a gene bank of serotype 5 Streptococcus pyogenes have been analyzed in detail. M proteins from partially purified bacteriophage lysates displayed precipitin lines of identity with a purified peptic extract of type 5 M protein (pep M5) in immunodiffusion assays. Immunoblot analyses of the M protein-positive lysates demonstrated that the cloned M protein component resided in five polypeptides with relative molecular weights of 57,900 (57.9K), 55.4K, 52.9K, 40.0K, and 32.6K. The hybrid lambda phage (lambda M5)-produced M protein contained immunoprotective epitopes; lambda M5 protein inhibited opsonization of type 5 streptococci by pep M5 antibodies, and antiserum raised against lambda M5 lysates opsonized type 5 streptococci. Each of the five antigenic polypeptides of the recombinant phage M protein also shared epitopes with human heart tissue, as demonstrated by the reactivity of immunoblots of lambda M5 antigens separated on sodium dodecyl sulfate gels with anti-pep M5 antibodies absorbed to and eluted from human heart sarcolemmal membranes. Moreover, antiserum raised against the lambda M5 lysates reacted with sarcolemmal membrane proteins with relative molecular weights of 200K, 59K, 55K, 53K, and 27K as determined by immunoblot analyses. These results demonstrate that the structural gene coding for type 5 streptococcal M protein which was inserted into lambda DNA expresses immunoprotective epitopes, some of which are shared with human heart tissue.

A procedure is described for the efficient transfer of fractionated bacterial lipopolysaccharide (LPS) from SDS-polyacrylamide gels to nitrocellulose filters, and its subsequent display by a peroxidase-linked antibody. The method is sensitive, and reveals and resolves high molecular weight LPS molecules having side chain lengths of up to and greater than 30 repeat units. It is also useful for the rapid analysis of LPS in bacterial outer membrane preparations.

Vaccine candidates against Shigella dysenteriae type 1, which is associated with the most severe cases of bacillary dysentery, were constructed. The rfp and rfb gene clusters, which code for S. dysenteriae 1 O antigen biosynthesis, were randomly integrated into either the chromosome or the virulence plasmid of the rough attenuated Shigella flexneri aroD strain SFL124-27 with a minitransposon carrying an arsenite resistance selection marker. The recombinant clones efficiently expressed the recombinant O antigen, exhibited a normal growth pattern, were able to invade and survive within eukaryotic cells to the same extent as the parental strain, and expressed the recombinant antigen within invaded cells. A clone was selected as the vaccine candidate, which was demonstrated to be immunogenic and safe in animal models, leading to 47% full protection and 53% partial protection against challenge with the wild-type strain.

Abstract: The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined.

Abstract: The biphenyl-mineralizing bacterium Burkholderia sp. strain LB400 also utilized 3-chloro-, 4-chloro-, 2,3′-dichloro- and 2,4′-dichlorobiphenyl for growth. By the attack of the initial enzyme a chlorine was eliminated dioxygenolytically from position 2 of one of the aromatic rings when hydrogens of both were substituted by chlorine. The strain mineralized 3-chloro- and 2,3′-dichlorobiphenyl via the central intermediate 3-chlorobenzoate through its chlorocatechol pathway enzymes, but excreted stoichiometric amounts of 4-chlorobenzoate from 4-chloro- and 2,4′-dichlorobiphenyl. These two compounds were mineralized by a co-culture of strain LB400 and a derivative of the (methyl-) benzoate-degrading strain Pseudomonas putida mt-2 (TOL). The complete degradation was achieved upon transfer of a cluster of at least five genes, encoding the regulated chlorocatechol pathway operon, from strain LB400 to strain mt-2. This transfer was demonstrated by the polymerase chain reaction.

Plasmid R6-5 contains a locus whose product inhibits induction of sfiA and prophage lambda in a recA441 mutant at 42 degrees C and in a recA+ host after treatment with nalidixic acid. This plasmidic SOS-inhibition locus (psi) is situated on an 8.1-kilobase DNA fragment near oriT, the origin of plasmid R6-5 conjugational transfer. Loss of the Psi function, resulting from the insertion of Tn3 into psi+, greatly reduced the synthesis of two proteins, designated PsiA (Mr 24,500) and PsiB (Mr 12,500). Using host cells in which there was an inactive LexA repressor, we found that Psi function does not act by interfering with the expression of the SOS pathway. The Psi function may affect the generation of an SOS signal. We postulate that during the course of evolution, the Psi function has been selected in some conjugative plasmids so as to permit them to transfer single-stranded DNA without generating an SOS signal.

Rhodococcus globerulus P6 (previously designated Acinetobacter sp. strain P6, Arthrobacter sp. strain M5, and Corynebacterium sp. strain MB1) is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. The genetic and biochemical analyses of the PCB catabolic pathway reported here have revealed the existence of a PCB gene cluster--bphBC1D--and two further bphC genes--bphC2 and bphC3--that encode three narrow-substrate-specificity enzymes (2,3-dihydroxybiphenyl dioxygenases) that meta cleave the first aromatic ring. None of the bphC genes show by hybridization homology to each other or to bphC genes in other bacteria, and the three bphC gene products have different kinetic parameters and sensitivities to inactivation by 3-chlorocatechol. This suggests that there exists a wide diversity in PCB meta cleavage enzymes.