Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.     

Novel hemoglobins to enhance microaerobic growth and substrate utilization in Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmp genes from P. aeruginosa, S. typhi, C. jejuni, K. pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A(600) 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A(600) 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D. radiodurans hemoprotein-expressing E. coli relative to the E. coli control carrying the parental plasmid without any hemoglobin gene.     

Obligatory coupling of colicin release and lysis in mitomycin-treated Col+ Escherichia coli

Previous work has shown that Escherichia coli K12 strains carrying the small, high copy number ColE2-P9 plasmid produce large amounts of colicin and then lyse and release colicin when grown in broth culture containing mitomycin C. Strains carrying the larger, low copy number ColIa-CA53 plasmid produced much less colicin and did not lyse or discharge more than 15% of their colicin when grown under the same conditions. Naturally-occurring Col+ strains and E. coli K12 derivatives carrying different Col plasmids could be classified either as ColE2+-like or ColIa+-like according to whether or not they produced large amounts of colicin and lysed and discharged colicin when grown in the presence of mitomycin, and also by the size and presumed copy number of the Col plasmid they carried. Strains carrying multiple copies of the cloned colicin Ia structural gene produced large amounts of colicin but did not lyse or release colicin when grown in the presence of mitomycin. This result rules out the possibility that high level accumulation of colicin is sufficient to cause lysis. Conditions were sought under which colicin Ia could be released from the producing cells. It was found that mitomycin-treated cultures of strains carrying both ColE2 and ColIa plasmids released both colicins when they lysed, although colicin Ia release occurred later than colicin E2 release. It was also noted that colicin Ia-laden cells released their colicin when diluted into fresh culture medium.     

Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae

In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAY101, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAY101 was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells.     

Colicin E2 production and release by Escherichia coli K12 and other Enterobacteriaceae

Previous work has shown that Escherichia coli K12 ColE2+ cells undergo a form of partial lysis and exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficient colicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPE content. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E. coli K12 as the model strain for studying the mechanisms of colicin release.     

Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells

When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12 strains of E. coli do not bind to sprouts under similar conditions. The roles of E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and exopolysaccharides in binding to sprouts were examined. An LPS mutant had no effect on the binding of the pathogenic strain. Cellulose synthase mutants showed a significant reduction in binding; colanic acid mutants were more severely reduced, and binding by poly-beta-1,6-N-acetylglucosamine (PGA) mutants was barely detectable. The addition of a plasmid carrying a cellulose synthase gene to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps biosynthesis genes had only a marginal effect on the binding of K-12 bacteria. However, the introduction of the same plasmid allowed Sinorhizobium meliloti and a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments. These results suggest that although multiple redundant protein adhesins are involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides required for binding are not redundant and each polysaccharide may play a distinct role. PGA, colanic acid, and cellulose were also required for biofilm formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7 to mammalian cells.     

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Molecular cloning of the structural genes of the Escherichia coli deo-operon in plasmid RSF2124]

A specialized phage lambda ddeo carrying the deo operon of Escherichia coli is analyzed by exposing the DNA to the specific restriction endonucleases EcoRI and BamHI. Using the lambda ddeo DNA fragment, obtained by digestion with BamHI and plasmid RSF2124 as a vehicle, the hybrid plasmid pAM1 carrying all the genes of the deo operon is constructed and cloned in E. coli cells. It is shown that the activity of thymidine phosphorylase in the strain AM061, which contains hybrid plasmid pAM1 is 30-fold greater than that in strains of E. coli with chromosomal localization of the deo operon.     

Evaluation of the interaction of phi X174 gene products E and K in E-mediated lysis of Escherichia coli.

Gene K of bacteriophage phi X174 was cloned, and its gene product was localized in the cell envelope of Escherichia coli. Compared with the sole expression of the phi X174 lysis gene E, the simultaneous expression of the K and E genes had no effect on scheduling of cell lysis. Therefore, a direct interaction of proteins E and K could be excluded. In contrast, phi X174 infection of a host carrying a plasmid expressing gene K resulted in a delayed lysis and an apparent increase in phage titer.     

Phage Folac: an Folac plasmid-dependent bacteriophage

By enriching sewage with Escherichia coli or Salmonella typhimurium strains harbouring the plasmid EDP208, a constitutive pilus-producing derivative of plasmid F olac, a phage was isolated which plated on these two organisms but not on isogenic strains without the plasmid. The phage was named F olac; it has a hexagonal outline with a diameter of 28 nm, contained RNA, was resistant to chloroform, and probably adsorbed preferentially to the sides of EDP208 pili very near the tip. Phage multiplication could be demonstrated on E. coli or S. typhimurium strains carrying the plasmid F olac, but an increase in titre did not occur on E. coli strains carrying plasmids of the F complex. Results of phage multiplication experiments on strains carrying the depressed pilus-producing plasmids R71 or TP224-Tc, which determine pili serologically related to those of EDP208, were inconclusive. Phage F olac was found to be serologically related to phage UA-6, another isolate specific for EDP208.     

Cloning and expression of genes encoding pheromone-inducible antigens of Enterococcus (Streptococcus) faecalis.

Fragments, generated by restriction enzyme digestion, of the 58-kilobase Enterococcus (Streptococcus) faecalis tetracycline resistance plasmid pCF10 were cloned and introduced into Escherichia coli and E. faecalis to characterize the pheromone-inducible conjugation system encoded by this plasmid. Western blot (immunoblot) analyses revealed that a 130-kilodalton (kDa) antigen, identical to the Tra130 antigen shown previously to be involved in pCF10-mediated pheromone-inducible surface exclusion, was produced by both bacterial hosts carrying the recombinant plasmid pINY1825 (cloned EcoRI C fragment). Both bacterial hosts carrying pINY1825 also produced various amounts of immunologically related 118- to 125-kDa antigens (designated pre-Tra130) that resembled antigens produced by E. faecalis cells carrying pCF10. An additional 150-kDa antigen, Tra150, probably involved in pheromone-induced cellular aggregation, was produced by Escherichia coli and E. faecalis hosts carrying pINY1801 (cloned EcoRI C and E fragments). The coding sequences for the Tra150 and Tra130 antigens were further localized in the TRA region of pCF10 by transposon insertion mutagenesis. Western blot analyses of the recombinant strains, and of strains carrying derivatives of pCF10 or various recombinant plasmids containing Tn5 or Tn917 insertions, suggested that the portion of pCF10 comprising the tra3 through -6 segments (previously defined by Tn917 insertional mutagenesis) contained several genes that are involved in regulating the synthesis of Tra130 and Tra150.     

One-step cloning system for isolation of bacterial lexA-like genes.

A system to isolate lexA-like genes of bacteria directly was developed. It is based upon the fact that the presence of a lexA(Def) mutation is lethal to SulA+ cells of Escherichia coli. This system is composed of a SulA- LexA(Def) HsdR- strain and a lexA-conditional killer vector (plasmid pUA165) carrying the wild-type sulA gene of E. coli and a polylinker in which foreign DNA may be inserted. By using this method, the lexA-like genes of Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa, and P. putida were cloned. We also found that the LexA repressor of S. typhimurium presented the highest affinity for the SOS boxes of E. coli in vivo, whereas the LexA protein of P. aeruginosa had the lowest. Likewise, all of these LexA repressors were cleaved by the activated RecA protein of E. coli after DNA damage. Furthermore, under high-stringency conditions, the lexA gene of E. coli hybridized with the lexA genes of S. typhimurium and E. carotovora but not with those of P. aeruginosa and P. putida.     

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.     

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.     

Regulation of ilvEDA expression occurs upstream of ilvG in Escherichia coli: additional evidence for an ilvGEDA operon.

A low-copy-number plasmid was prepared that contained the entire ilv gene cluster of Escherichia coli. The introduction of an ilvO mutation allowed the ilvG gene of the plasmid to be expressed and imparted valine resistance to strains carrying it. Insertion of Tn10 into the ilvG gene of the plasmid resulted in a strong polar effect on ilv genes E, D, and A. Replacement of a region of ilv deoxyribonucleic acid between two KpnI sites on the high-copy-number plasmid carrying the entire ilv gene cluster with a KpnI fragment carrying an ilv-lac fusion but not extending into the ilv-specific control region resulted in a plasmid expressing the lacZ gene under ilv control when the fusion had been inserted in its normal orientation but not when it had been inserted in the opposite orientation. These experiments indicate that ilv-specific control over ilvE, ilvD, and ilvA expression is dependent on these genes being continguous with deoxyribonucleic acid that lies upstream of ilvG. The results also add further support to the concept of an ilvGEDA operon in E. coli.     

A gene between polA and glnA retards growth of Escherichia coli when present in multiple copies: physiological effects of the gene for spot 42 RNA.

We have isolated the single gene for spot 42 RNA of Escherichia coli on a 20-kilobase DNA fragment. Physical characterization of this cloned DNA fragment showed that it is homologous to a region at 86 min on the genetic map and extends from the 23S to 5S rRNA coding region of rrnA to the coding region of glnA, the gene for glutamine synthetase. Other genes included on this cloned DNA fragment are polA, ntrC (glnG), and ntrB (glnL). E coli cells transformed with a multicopy plasmid clone of the gene for spot 42 RNA had about a 10-fold increase in the amount of spot 42 RNA they contained. The amount of 6S RNA in these cells was increased about twofold, although the gene for 6S RNA was not located on this plasmid or on the larger 20-kilobase fragment. Presence of this multicopy plasmid also affected the growth of cells. The generation time was increased under a variety of growth conditions, especially when cells were grown in medium with succinate as the carbon source. In addition, some strains of E. coli which have multicopy plasmids carrying the gene for spot 42 RNA were unable to respond normally to a shift into richer medium: upon upshift from minimal glucose to LB broth or minimal glucose plus 1% Casamino Acids, there was a 3- to 4-h lag before the culture adapted to the new medium. More than 90% of the cells in such cultures stopped dividing, although they remained viable. The plating efficiency of minimal-glucose-grown cells was 100-fold less on rich media than on minimal glucose medium. One revertant was isolated which regained the phenotype of pBR322-transformed cells. Analysis of this strain showed that the plasmid it contained had an insertion of an IS1 element into the 5' end of the coding region for the gene for spot 42 RNA.     

Transgenosis with participation of plasmid RP1; indications of the presence of a "composit plasmid" in an interspecies hybrid of Escherichia coli]

One of the transconjugants (1-7) obtained by the authors earlier in the conjugation of Escherichia coli J-62 with Pseudomonas aeruginosa 1822, besides the plasmic RP1 has acquired the ability to grow without proline and tryptophan. The detailed analysis has shown that in the conjugation of the transconjugant 1-7 with different strains of E. coli the plasmic RP1 and chromosomal genes were transmitted together, but in transduction--by means of bacteriophage P1, independently of each other. The fertility was found only in the transductants carrying the plasmid RP1. This suggests that in the intergeneric conjugations the transmission of chromosomal genes may occur without any firm link with the plasmid (as in the case of "aggregated plasmids"). In E. coli cells these chromosomal fragments of Ps. aeruginosa apparently formed small nontransmissible replicons.