Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae

D-Galactan I is an O-antigenic polymer with the repeat unit structure [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of D-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of D-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The D-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into D-galactan I biosynthesis by the bifunctional wbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of D-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the D-galactan I polymer requires WbbM for Galp transferase, together with Galf transferase activity provided by WbbO. Comparison of the biosynthetic pathways for D-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.     

Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida.

Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression, localization, and functional analysis of polychlorinated biphenyl degradation genes cbpABCD of Pseudomonas putida.

Genes of Pseudomonas putida strains that are capable of degrading polychlorinated biphenyls were cloned in the plasmid vector pUC19. The resultant hybrid plasmid, pAW6194, contained cbpABCD genes on a 9.0-kb DNA fragment that was necessary for the catabolism of polychlorinated biphenyls. These genes were further subcloned on an 8.0-kb HindIII fragment of pAW540. Degradation of 3-chlorobiphenyl, 2,4-dichlorobiphenyl, and 2,4,5-trichlorobiphenyl into a chloro derivative of benzoic acid was found in Escherichia coli harboring chimeric plasmid pAW540. Expression of cbpA (biphenyl dioxygenase, 6.2 U/mg of protein) and cbpC (3-phenylcatechol dioxygenase, 611.00 U/mg of protein) genes was also found in E. coli containing the hybrid plasmid pAW540. These enzyme activities were up to 10-fold higher than those found in P. putida OU83. These results led us to conclude that cbpABCD genes of P. putida OU83 were encoded on cloned DNA and expressed in E. coli. Whether the expression of cbpABCD genes of P. putida OU83 was driven by its own promoters located on the cloned DNA or by the lacZ promoter of pUC19 was examined by subcloning a 8.0-kb DNA fragment encoding the cbpABCD genes, in both orientations, in the HindIII site of the promoter probe vector pKK232-8. The resulting recombinant plasmids, pAW560 and pAW561, expressed cbpABCD genes and conferred chloramphenicol resistance only in E. coli harboring pAW560, indicating that the expression of chloramphenicol acetyltransferase is independent of cbpABCD gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

A UDP-HexNAc:polyprenol-P GalNAc-1-P transferase (WecP) representing a new subgroup of the enzyme family

The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the donor substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.     

Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei.

The purL gene from Lactobacillus casei, encoding phosphoribosylformylglycinamidine synthase II involved in the de novo synthesis of purines, was cloned and sequenced. The putative purL product of 741 amino acids (M(r) of 79,575) shows 25% and 53% identity to the homologous enzymes from Escherichia coli and Bacillus subtilis, respectively. In addition, partial sequences of two other pur genes (purQ and purF) and a possible third gene (purC) were obtained. All these genes are organized in an operon similar to that of B. subtilis. In contrast, the corresponding genes from E. coli and Salmonella typhimurium are scattered through the genome.     

Identification of restriction fragments from two cryptic Clostridium butyricum plasmids that promote the establishment of a replication-defective plasmid in Bacillus subtilis

Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6.05 kbp) and pCB102 (7.8 kbp). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E. coli. A 3.3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+ and Rec- strains of B. subtilis. Plasmid pRB1, a representative chimaera carrying only the 3.3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2.0 kbp Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome.     

Replication Mechanism and Sequence Analysis of the Replicon of pAW63, a Conjugative Plasmid from Bacillus thuringiensis

A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti.     

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

DNA polymerase III of Enterococcus faecalis: expression and characterization of recombinant enzymes encoded by the polC and dnaE genes

Enterococcus faecalis (Ef) dnaE and polC, the respective genes encoding the DNA replication-specific DNA polymerase III E and DNA polymerase III C, were cloned and engineered for expression in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Each gene expressed a catalytically active DNA polymerase of the expected molecular weight. The recombinant polymerases were purified and each was characterized with respect to catalytic properties, inhibitor sensitivity, and recognition by specific antibody raised against the corresponding DNA polymerase III of the model Gram-positive (Gr(+)) organism, Bacillus subtilis (Bs). In conclusion, the properties of each Enterococcus polymerase enzymes were similar to those of the respective B. subtilis enzymes.     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Identification and localization of 3-phenylcatechol dioxygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase genes of Pseudomonas putida and expression in Escherichia coli

The bphC and bphD genes of Pseudomonas putida involved in the catabolism of polychlorinated biphenyls or biphenyl were identified, localized, and studied for expression in Escherichia coli. This was achieved by cloning a 2.4-kilobase (kb) DNA fragment of recombinant cosmid pOH101 into HindIII site of pUC plasmids downstream of a lacZ promoter and measuring the enzyme activities of 3-phenylcatechol dioxygenase (3-PDase; a product of bphC) and the meta-cleavage product 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (a product of bphD). The amount of 3-PDase produced in E. coli was about 20 times higher than that of the enzyme produced by the parent, P. putida. Determination of expression of the bphC and bphD genes through their own promoter sequences or by using the lacZ promoter of pUC plasmids was done by cloning the DNA that encodes bphC and bphD genes in a HindIII site of a promoter selection vector (pKK232-8) upstream of the gene for chloramphenicol acetyltransferase (CAT). The recombinant plasmid (pAW787) constructed by inserting the 2.4-kb DNA in pKK232-8 expressed both 3-PDase and CAT activities. Another hybrid construct (pAW786) in which the DNA insert was cloned in the opposite orientation lacked CAT activity but produced normal amounts of 3-PDase activity. On the basis of these results, we suggest that the bphC and bphD genes were expressed by using promoter sequences that are independent of the promoter that expresses CAT activity in E. coli. The locations of the bphC and bphD genes were determined by insertional inactivation of the open reading frames of structural genes bphC and bphD by Tn5 mutagenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY

A 3.5-kb HindIII DNA fragment containing the secY gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis secY homologue was composed of 423 amino acids and its molecular weight was calculated to be 46,300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis SecY homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.     

Identification of enzymes acting on alpha-glycated amino acids in Bacillus subtilis

We have characterized the Bacillus subtilis homologs of fructoselysine 6-kinase and fructoselysine-6-phosphate deglycase, two enzymes that specifically metabolize the Amadori compound fructose-epsilon-lysine in Escherichia coli. The B. subtilis enzymes also catalyzed the phosphorylation of fructosamines to fructosamine 6-phosphates (YurL) and the conversion of the latter to glucose 6-phosphate and a free amino acid (YurP). However, their specificity was totally different from that of the E. coli enzymes, since they acted on fructoseglycine, fructosevaline (YurL) or their 6-phosphoderivatives (YurP) with more than 30-fold higher catalytic efficiencies than on fructose-alpha-lysine (6-phosphate). These enzymes are therefore involved in the metabolism of alpha-glycated amino acids.     

Comparative action of glyphosate as a trigger of energy drain in eubacteria.

Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa, each possessing a 5-enolpyruvylshikimate 3-phosphate synthase that is sensitive to inhibition by glyphosate [N-(phosphonomethyl)glycine], provide a good cross-section of organisms exemplifying the biochemical diversity of the aromatic pathway targeted by this potent antimicrobial compound. The pattern of growth inhibition, the alteration in levels of aromatic-pathway enzymes, and the accumulation of early-pathway metabolites after the addition of glyphosate were distinctive for each organism. Substantial intracellular shikimate-3-phosphate accumulated in response to glyphosate treatment in all three organisms. Both E. coli and P. aeruginosa, but not B. subtilis, accumulated near-millimolar levels of shikimate-3-phosphate in the culture medium. Intracellular backup of common-pathway precursors of shikimate-3-phosphate was substantial in B. subtilis, moderate in P. aeruginosa, and not detectable in E. coli. The full complement of aromatic amino acids prevented growth inhibition and metabolite accumulation in E. coli and P. aeruginosa where amino acid end products directly control early-pathway enzyme activity. In contrast, the initial prevention of growth inhibition in the presence of aromatic amino acids in B. subtilis was succeeded by progressively greater growth inhibition that correlated with rapid metabolite accumulation. In B. subtilis glyphosate can decrease prephenate concentrations sufficiently to uncouple the sequentially acting loops of feedback inhibition that ordinarily link end product excess to feedback inhibition of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by prephenate. The consequential unrestrained entry is an energy-rich substrates into the aromatic pathway, even in the presence of aromatic amino acid end products, is an energy drain that potentially accounts for the inability of end products to fully reverse glyphosate inhibition in B. subtilis. Even in E. coli after glyphosate inhibition and metabolite accumulation were allowed to become fully established, a transient period where end products were capable of only partial reversal of growth inhibition occurred. The distinctive metabolism produced by dissimilation of different carbon sources also profound effects upon glyphosate sensitivity.     

Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis

The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.     

Production of xylitol by metabolically engineered strains of Bacillus subtilis

Xylitol-phosphate dehydrogenase (XPDH) genes from several Gram-positive bacteria were isolated and expressed in Bacillus subtilis. The substrate specificities of the recombinant XPDH enzymes were compared and it was found that the XPDH enzymes of Lactobacillus rhamnosus and Clostridium difficile had the highest selectivity towards D-xylulose 5-phosphate. Expression of these two XPDH enzymes in D-ribulose and D-xylulose producing B. subtilis strain resulted in strains of B. subtilis capable of converting D-glucose into xylitol at around 23% yield.     

Cloning and sequencing of the beta-lactamase I gene of Bacillus cereus 5/B and its expression in Bacillus subtilis.

The beta-lactamases of Bacillus cereus have attracted interest because they are secreted efficiently, because multiple enzymes are frequently present, and because their regulation has unusual features. beta-Lactamase I of strain 5/B is produced constitutively at a high level, and the exoenzyme appears to be several thousand daltons larger than the corresponding product of strain 569/H. We have cloned the gene for 5/B beta-lactamase I in Escherichia coli and B. subtilis and have sequenced the structural portion and the regulatory regions. The 5/B enzyme is produced at a low level in E. coli RR1(pRWY200) and remains cellbound. In B. subtilis it is formed in large amounts, and over 90% of it is released into the medium. There is a large degree of homology between the promoter and leader peptide regions of the 5/B and 569/H genes; both utilize UUG as the translation initiation codon (P. S. F. Mézes, R. W. Blacher, and J. O. Lampen, (J. Biol. Chem. 260:1218-1223, 1985). Although there are significant differences in the peptide segment where processing would be expected to occur, the NH2 terminus of the major 5/B product from B. subtilis BD170(pRWY215) is His-44, which is the same as the NH2 terminus of the major 569/H product from B. subtilis BD170(pRWM5).