A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase

Using DNA obtained from the metagenome of an anaerobic digestor in a waste water treatment plant, we constructed a gene library cloned in the wide host-range cosmid pLAFR3. One cosmid enabled Rhizobium leguminosarum to grow on ethanol as sole carbon and energy source, this being due to the presence of a gene, termed adhEMeta. The AdhEMeta protein most closely resembles the AdhE alcohol dehydrogenase of Clostridium acetobutylicum, where it catalyses the formation of ethanol and butanol in a two-step reductive process. However, cloned adhEMeta did not confer ethanol utilization ability to Escherichia coli or to Pseudomonas aeruginosa, even though it was transcribed in both these hosts. Further, cell-free extracts of E. coli and R. leguminosarum containing cloned adhEMeta had butanol and ethanol dehydrogenase activities when assayed in vitro. In contrast to the well-studied AdhE proteins of C. acetobutylicum and E. coli, the enzyme specified by adhEMeta is not inactivated by oxygen and it enables alcohol to be catabolized. Cloned adhEMeta did, however, confer one phenotype to E. coli. AdhE- mutants of E. coli fail to ferment glucose and introduction of adhEMeta restored the growth of such mutants when grown under fermentative conditions. These observations show that the use of wide host-range vectors enhances the efficacy with which metagenomic libraries can be screened for genes that confer novel functions.     

Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300

Abstract Expression of the cloned PhiX174 gene E causes lysis of the Gram-negative bacterium Escherichia coli , which led to the proposal that a two-membrane system is necessary for the protein E lysis function. Gene E was cloned in an E. coli/Bacillus subtilis shuttle vector and expressed in the Gram-positive bacterium Staphylococcus carnosus TM300. Regulated gene E expression had a lethal effect on S. carnosus ; however, no lysis was detected, lending support to the hypothesis.     

Role of autolysins in the EDTA-induced lysis of Pseudomonas aeruginosa

Abstract A DNA fragment containing the genes secE, nusG and rplK of Staphylococcus carnosus was cloned using the Escherichia coli rplK gene as a probe. The S. carnosus secE homologue encodes a protein of 65 amino acid residues which is homologous to the carboxyl-terminal region of the E. coli SecE protein. The S. carnosus SecE polypeptide which, in contrast to the E. coli SecE protein, contains only one putative transmembrane segment, could fully replace the E. coli SecE protein in two different secE mutants. These results strongly suggest that the identified secE gene encodes an important component of the S. carnosus protein export apparatus.     

Cloning,expression, and functional characterization of the Mycobacterium tuberculosis secA gene

To better understand the protein secretion mechanisms involved in the growth and pathogenesis of Mycobacterium tuberculosis, we examined the secA gene from M. tuberculosis (tbsecA; cosmid sequence accession No. z95121.gb_ba). We generated plasmids containing the full-length tbsecA gene or a fusion containing the 5' sequence from the M. tuberculosis secA gene and the remainder from the Escherichia coli secA gene and evaluated the ability of each construct to complement the defective SecA protein in E. coli MM52ts when grown at the non-permissive temperature. The full-length tbsecA gene was unable to compensate for the temperature-sensitive defect, whereas E. coli MM52ts that has been transformed with plasmid pMF8TB226 containing a chimeric secA gene was able to grow at 42 degrees C. This work confirms that the topography of SecA and its ATP binding sites are highly conserved, whereas its membrane insertion domains are species specific.     

Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA(218)) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 x 10(4) E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23 degrees C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA(218) fusion protein at 23 and 30 degrees C but unexpectedly not at 37 degrees C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA(218) was detected only in the L448F mutant (AE104F) at 23 degrees C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA(218) fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37 degrees C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.     

Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus

The gene which encodes the mannitol-specific enzyme III (EIIImtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp Dde I fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.     

Gene cloning and expression of the Pseudomonas aeruginosa periplasmic phosphate-binding protein

Abstract The pstS gene, encoding the Pseudomonas aeruginosa phosphate-binding protein, was cloned onto a cosmid vector into Escherichia coli , and localized by subcloning, mapping the insertion site of Tn 501 in a P. aeruginosa pstS ::Tn 501 mutant, and hybridization to an oligonucleotide pool synthesized according to the aminoterminal amino acid sequence of the purified protein. The cloned pstS gene was transferred to P. aeruginosa pstS mutants. The P. aeruoginosa phosphate-binding protein was also expressed and secreted into the periplasm of E. coli pstS mutants.     

Genetic and molecular analyses of Escherichia coli K1 antigen genes

The plasmid pSR23, composed of a 34-kilobase E. coli chromosomal fragment inserted into the BamHI site of the pHC79 cosmid cloning vector, contains genes encoding biosynthesis of the K1 capsular polysaccharide. Deletions, subclones, and Tn5 insertion mutants were used to localize the K1 genes on pSR23. The only deletion derivative of pSR23 that retained the K1 phenotype lacked a 2.7-kilobase EcoRI fragment. Subclones containing HindIII and EcoRI fragments of pSR23 did not produce K1. Cells harboring pSR27, a subclone containing a 23-kilobase BamHI fragment, synthesized K1 that was not detectable extracellularly. Six acapsular Tn5 insertion mutants of three phenotypic classes were observed. Class I mutants synthesized K1 only when N-acetylneuraminic acid (NANA) was provided in the medium. Reduced amounts of K1 were detectable in cell extracts of class II mutants. Class III mutants did not produce detectable K1 in either extracts or when cells were provided exogenous NANA. All mutants had sialyltransferase activity. Analysis in the E. coli minicell system of proteins expressed by derivatives of pSR23 identified a minimum of 12 polypeptides, ranging in size from 18,000 to 80,000 daltons, involved in K1 biosynthesis. The 16-kilobase coding capacity required for the proteins was located in three gene clusters designated A, B, and C. We propose that the A cluster contains a NANA operon of two genes that code for proteins with apparent molecular weights of 45,000 and 50,000. The A region also includes a 2-kilobase segment involved in regulation of K1 synthesis. The B region encoding five protein species appears responsible for the translocation of the polymer from its site of synthesis on the cytoplasmic membrane to the cell surface. The C region encodes four protein species. Since the three gene clusters appear to be coordinately regulated. we propose that they constitute a kps regulon.     

Isolation of Escherichia coli mutants defective in uptake of molybdate.

For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.     

Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins

Saccharomyces cerevisiae Cdc7 kinase is required for initiation of S phase, and its kinase activity, which is positively regulated by Dbf4 protein, reaches maximum at the G1/S boundary. In this study, we constructed Cdc7 point mutants (T281E, T281A, D182N, D163N, and T167E) and examined the effect of each mutant on growth. All the mutants lost the ability to complement temperature-sensitive growth of cdc7(ts) mutants at a low protein level, whereas T281A (putative target of phosphorylation) and T167E (residue involved in substrate recognition) restored the growth of cdc7(ts) when overproduced to a high level. Three putative kinase-negative mutants (T281E, D182N, and D163N) inhibited growth when overexpressed in a wild-type strain. Analyses of DNA content and morphology revealed that most cells were arrested as dumbbells with 1C DNA, indicative of a block in the G1 to S transition. This growth inhibition was suppressed by co-overexpression of the wild-type Cdc7 or Dbf4 protein. Furthermore, deletion of the Dbf4 protein-binding region in each Cdc7 mutant resulted in loss of growth inhibitory effect. Thus, dominant-negative effects of T281E, D182N, and D163N on growth can be best explained by inactivation of the wild-type Cdc7 function through titration of Dbf4 by these inactive kinases. Our results are consistent with the notion that association of Dbf4 with Cdc7 is essential for the G1 to S transition in S. cerevisiae. Received: 17 September 1996 / Accepted: 6 January 1997     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.     

Sequence analysis of insecticidal genes from Xenorhabdus nematophilus PMFI296

Three strains of Xenorhabdus nematophilus showed insecticidal activity when fed to Pieris brassicae (cabbage white butterfly) larvae. From one of these strains (X. nematophilus PMFI296) a cosmid genome library was prepared in Escherichia coli and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in E. coli (50% lethal concentration [LC(50)] of 2 to 6 microg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (xptA1, -A2, -B1, -C1, and -D1) showed homology with up to 49% identity to insecticidal toxins identified in Photorhabdus luminescens, and also a smaller gene (chi) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes xptA2, xptD1, and chi were not important for the expression of insecticidal activity toward P. brassicae. One gene (xptA1) was found to be central for the expression of activity, and the genes xptB1 and xptC1 were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this E. coli clone. Although multiple genes may be needed for full activity, E. coli cells expressing the xptA1 gene from the bacteriophage lambda P(L) promoter were shown to have insecticidal activity (LC(50) of 112 microg of total protein/g of diet). This is contrary to the toxin genes identified in P. luminescens, which were not insecticidal when expressed individually in E. coli. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the E. coli clone expressing xptA1. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of X. nematophilus PMFI296.     

Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins

Saccharomyces cerevisiae Cdc7 kinase is required for initiation of S phase, and its kinase activity, which is positively regulated by Dbf4 protein, reaches maximum at the G1/S boundary. In this study, we constructed Cdc7 point mutants (T281E, T281A, D182N, D163N, and T167E) and examined the effect of each mutant on growth. All the mutants lost the ability to complement temperature-sensitive growth of cdc7(ts) mutants at a low protein level, whereas T281A (putative target of phosphorylation) and T167E (residue involved in substrate recognition) restored the growth of cdc7(ts) when overproduced to a high level. Three putative kinase-negative mutants (T281E, D182N, and D163N) inhibited growth when overexpressed in a wild-type strain. Analyses of DNA content and morphology revealed that most cells were arrested as dumbbells with 1C DNA, indicative of a block in the G1 to S transition. This growth inhibition was suppressed by co-overexpression of the wild-type Cdc7 or Dbf4 protein. Furthermore, deletion of the Dbf4 protein-binding region in each Cdc7 mutant resulted in loss of growth inhibitory effect. Thus, dominant-negative effects of T281E, D182N, and D163N on growth can be best explained by inactivation of the wild-type Cdc7 function through titration of Dbf4 by these inactive kinases. Our results are consistent with the notion that association of Dbf4 with Cdc7 is essential for the G1 to S transition in S. cerevisiae.     

Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.     

Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis

A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.     

Cloning of an insecticidal toxin gene of Bacillus thuringiensis subsp. tenebrionis and its expression in Escherichia coli cells

Abstract A structural gene of a crystal protein toxic for coleoptera larvae was cloned from plasmid DNA of Bacillus thuringiensis subsp. tenebrionis (BTT). The DNA was partially digested with restriction enzyme Bam HI and fragments were inserted into cosmid pHC79. In Western blot analysis extracts from infected Escherichia coli cells revealed expression of the BTT crystal protein in antibiotic-resistant cells. Cell lysates from a selected E. coli clone were toxic for larvae of the Colorado potato beetle ( Leptinotarsa decemlineata ). The electrophoretic mobility in SDS gels of crystal protein from E. coli cells was 68 kDa and 74 kDa as observed for BTT-toxin in B. thuringiensis extracts. The cosmids obtained were unstable during cellular propagation. The deletion product still carried the δ-endotoxin gene.