Expression of the Escherichia coli pfkA gene in Alcaligenes eutrophus and in other gram-negative bacteria.

The Escherichia coli pfkA gene has been cloned in the non-self-transmissible vector pVK101 from hybrid plasmids obtained from the Clarke and Carbon clone bank, resulting in the plasmids pAS300 and pAS100; the latter plasmid also encoded the E. coli tpi gene. These plasmids were transferred by conjugation to mutants of Alcaligenes eutrophus which are unable to grow on fructose and gluconate due to lack of 2-keto-3-deoxy-6-phosphogluconate aldolase activity. These transconjugants recovered the ability to grow on fructose and harbored pAS100 or pAS300. After growth on fructose, the transconjugants contained phosphofructokinase at specific activities between 0.73 and 1.83 U/mg of protein, indicating that the E. coli pfkA gene is readily expressed in A. eutrophus and that the utilization of fructose occurs via the Embden-Meyerhof pathway instead of the Entner-Doudoroff pathway. In contrast, transconjugants of the wild type of A. eutrophus, which are potentially able to catabolize fructose via both pathways, grew at a decreased rate on fructose and during growth on fructose did not stably maintain pAS100 or pAS300. Indications for a glycolytic futile cycling of fructose 6-phosphate and fructose 1,6-bisphosphate are discussed. Plasmid pA 100 was also transferred to 14 different species of gram-negative bacteria. The pfkA gene was expressed in most of these species. In addition, most transconjugants of these strains and of A. eutrophus exhibited higher specific activities of triosephosphate isomerase than did the corresponding parent strains.     

traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.     

Biotinylation facilitates the uptake of large peptides by Escherichia coli and other gram-negative bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

Induction of the alkA gene of Escherichia coli in gram-negative bacteria.

A broad-host-range plasmid containing a fusion of the alkA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria--33 species belonging to 19 genera--to study the induction of expression of the alkA gene by alkylating agents. The bacteria included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae, and Azotobacteraceae. Results obtained show that all bacteria tested, except Aeromonas hydrophila, Agrobacterium tumefaciens, Hafnia alvei, Rhizobium meliloti, Salmonella enteritidis, Xanthomonas campestris, and those of the genus Rhodobacter, are able to induce the alkA gene of E. coli in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. All these data indicate that the adaptive response to alkylating agents is present in bacterial species of several families and that the Ada box sequence must be widely conserved.     

Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeat sequences originally discovered in Escherichia coli and Salmonella typhimurium. We have physically mapped these sequences in the E. coli genome by using Southern hybridization of an ordered phage bank of E. coli (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) with generic REP probes derived from the REP consensus sequence. The set of REP probe-hybridizing clones was correlated with a set of clones expected to contain REP sequences on the basis of computer searches. We also show that a generic REP probe can be used in Southern hybridization to analyze genomic DNA digested with restriction enzymes to determine genetic relatedness among natural isolates of E. coli. A search for these sequences in other members of the family Enterobacteriaceae shows a consistent correlation between both the number of occurrences and the hybridization strength and genealogical relationship.     

Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli.

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli

The cer –Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat. Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA. The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity. This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint. We describe here the characterization of a novel class of 'conditionally constrained' multimer resolution sites whose properties support this hypothesis. In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen. A mutant ArgR derivative (ArgR110), which is defective in cer -mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site. This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution. When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained. Constraint was restored by the insertion of a full helical turn.     

Immunological investigation of the distribution of cytochromes related to the two terminal oxidases of Escherichia coli in other gram-negative bacteria.

Monospecific antibodies were raised against the two terminal oxidase complexes of the aerobic respiratory chain of Escherichia coli. These are the cytochrome d and cytochrome o complexes. The antibodies were used to check for the occurrence of cross-reactive antigens in membrane preparations from a variety of gram-negative bacteria by rocket immunoelectrophoresis and immunoblotting techniques. With these criteria, proteins closely related to the cytochrome d complex of E. coli appeared to be widely distributed. Among the strains containing cytochrome d-related material were Serratia marcescens, Photobacterium phosphoreum, Salmonella typhimurium, Klebsiella pneumoniae, and Azotobacter vinelandii. The data suggest that the d-type terminal oxidase in many of these strains is associated in a complex with b-type and a1-type cytochromes, as has been found to be the case in E. coli. K. pneumoniae and S. typhimurium were also shown to have material cross-reactive to the E. coli cytochrome o complex.     

Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes.

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.     

Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compilation and analysis of Escherichia coli promoter DNA sequences.

The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence.     

Major heat-modifiable outer membrane protein in gram-negative bacteria: comparison with the ompA protein of Escherichia coli.

The outer membranes of several strains of Escherichia coli, other enteric bacteria, and a variety of nonenteric gram-negative bacteria all contain a major heat-modifiable protein similar to the OmpA protein of E. coli K-12. The heat-modifiable proteins from these bacteria resemble the K-12 protein in molecular weight, in preferential release from the outer membrane by sodium dodecyl sulfate in the presence of Mg2+, and in characteristic cleavage by proteases to yield a smaller fragment which remains membrane bound. Antiserum directed against the K-12 protein precipitated the heat-modifiable protein from all strains of Enterobacteriaceae, and chemical comparison by isoelectric focusing, cyanogen bromide cleavage profiles, and proteolytic peptide analysis indicated that the proteins from the various enteric bacteria were nearly identical in primary structure. The heat-modifiable proteins from bacteria phylogenically distant from E. coli shared many of the properties of the E. coli protein but were chemically distinct. Thus, it appears that the structure (and, presumably, the function) of the heat-modifiable protein of gram-negative bacteria is strongly conserved during evolution.