Paul Luther Skatrud

Genome of the Bacterium Streptococcus pneumoniae Strain R6

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum

The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe. The protein coding region of the P. chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum.

Cloning, disruption and sequence of the gene encoding yeast C-5 sterol desaturase

The ERG3 gene from Saccharomyces cerevisiae has been cloned by complementation of an erg3-2 mutation. ERG3 is the putative gene encoding the C-5 sterol desaturase required for ergosterol biosynthesis. The functional gene has been localized on a 2.5-kb HindIII-BamHI fragment containing an open reading frame comprising 365 amino acids. Gene disruption resulting from a deletion/substitution demonstrates that ERG3 is not essential for cell viability or the sparking function.

Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1 and AflMDR1 encode proteins of molecular weights 148,000 and 143,000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85,000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.

The role of ABC transporters from Aspergillus nidulans in protection against cytotoxic agents and in antibiotic production.

Abstract This paper describes the characterization of atrC and atrD (ABC transporters C and D), two novel ABC transporter-encoding genes from the filamentous fungus Aspergillus nidulans, and provides evidence for the involvement of atrD in multidrug transport and antibiotic production. BLAST analysis of the deduced amino acid sequences of AtrCp and AtrDp reveals high homology to ABC transporter proteins of the P-glycoprotein cluster. AtrDp shows a particularly high degree of identity to the amino acid sequence of AfuMdr1p, a previously characterized ABC transporter from the human pathogen A. fumigatus. Northern analysis demonstrates an increase in transcript levels of atrC and atrD in fungal germlings upon treatment with natural toxic compounds and xenobiotics. The atrC gene has a high constitutive level of expression relative to atrD, which suggests its involvement in a metabolic function. Single knock-out mutants for atrC and atrD were generated by gene replacement using pyrG from A. oryzae as a selectable marker. ΔatrD mutants display a hypersensitive phenotype to compounds such as cycloheximide, the cyclosporin derivative PSC 833, nigericin and valinomycin, indicating that AtrDp is involved in protection against cytotoxic compounds. Energy-dependent efflux of the azole-related fungicide fenarimol is inhibited by substrates of AtrDp (e.g. PSC 833, nigericin and valinomycin), suggesting that AtrDp plays a role in efflux of this fungicide. Most interestingly, ΔatrD mutants display a decrease in penicillin production, measured indirectly as antimicrobial activity against Micrococcus luteus. These results suggest that ABC transporters may be involved in secretion of penicillin from fungal cells.

Efficient integrative transformation of Cephalosporium acremonium

SummaryA hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 by sequence of Cephalosporium acremonium DNA next to the 5′ end of a bacterial open reading frame, HPTorf. The sequence was obtained as an 850 by NcoI restriction fragment from the 5′ noncoding region of the C. acremonium isopenicillin N synthetase (IPNS) gene. The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT). Plasmids that contained IPNSp/HPTorf transformed C. acremonium to a stably maintained hygromycin B resistant phenotype. Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants. The number of transformants per μg of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence. Plasmids with the promoterless HPTnorf and plasmids with a truncated S. cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C. acremonium at equivalent low frequencies.Transformation of C. acremonium with linearized plasmid DNA produced at least 2–3 fold more transformants than the corresponding circular molecule. Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells.Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C. acremonium ribosomal DNA (rDNA), or a fragment of C. acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S. cerevisiae, or putative transcriptional tennination/polyadenylation signals from the IPNS gene. These plasmids transformed C. acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.

An electrophoretic molecular karyotype for an industrial strain of Cephalosporium acremonium

An electrophoretic molecular karyotype has been established for an industrial strain of Cephalosporium acremonium using transverse alternating field electrophoresis. Eight chromosome bands were detected in gently prepared DNA samples. The size of the chromosomes ranged from approx. 1700 kb up to greater than 4000 kb. The total genomic content for this strain of C. acremonium is at least 22,500 kb. Hybridization analyses revealed that two key genes involved in cephalosporin C biosynthesis are not physically linked to one another. The isopenicillin N synthetase gene (pcbC) resides on chromosome (chr.) VI while the deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase gene (cefEF) resides on chr. II. The ribosomal RNA genes were located on chr. VII, while the beta-isopropylmalate dehydrogenase gene (LEU2) was found to be linked to the pcbC gene on chr. VI.

Cloning and expression of a hybrid Streptomyces clavuligerus cefE gene in Penicillium chrysogenum

SummaryA hybrid cefE gene was constructed by juxtaposing promoter sequences from the Penicillium chrysogenum pcbC gene to the open reading frame of the Streptomyces clavuligerus cefE gene. In S. clavuligerus the cefE gene codes for the enzyme penicillin N expandase [also known as deacetoxycephalosporin C synthetase (DAOCS)]. To insert the hybrid cefE gene into P. chrysogenum the vector pPS65 was constructed; pPS65 contains the hybrid cefE gene and the Aspergillus nidulans amdS gene. The amdS gene encodes acetamidase and provides for dominant selection in P. chrysogenum. Protoplasts of P. chrysogenum were transformed with pPS65 and selected for the ability to grow on acetamide medium. Extracts of cells cultivated in penicillin production medium were analyzed for penicillin N expandase activity. Penicillin N expandase activity was detected in approximately one-third of the transformants tested. Transformants WG9-69C-01 and WG9-61L-03 had the highest specific activities of penicillin N expandase: 4.3% and 10.3%, respectively, relative to the amount of penicillin N expandase in S. clavuligerus. Untrasformed P. chrysogenum exhibited no penicillin N expandase activity. Analysis of the penicillin V titer revealed that WG9-61L-03 produced titers equal to that of the recipient strain while the amount of penicillin V produced in WG9-69C-01 was reduced by five fold.

CLONING AND CHARACTERIZATION OF CNEMDR1: A CRYPTOCOCCUS NEOFORMANS GENE ENCODING A PROTEIN RELATED TO MULTIDRUG RESISTANCE PROTEINS

CneMDR1, a gene encoding a protein related to several eukaryotic multidrug resistance (MDR) proteins, was identified, cloned, and characterized from a clinical isolate of Cryptococcus neoformans (Cn) (strain M1-106). Polymerase chain reaction (PCR) amplification of a DNA region encompassing conserved motifs of other MDR-like proteins was initially used to identify and clone CneMDR1. Analysis of the corresponding cDNA revealed an open reading frame punctuated by 16 introns. CneMDR1 encoded a protein (CNEMDR1) containing 1408 amino acids (aa) with a predicted mass of approximately 152kDa. Protein structure predictions suggested the presence of two putative 6-transmembrane (TM) domains as well as two ATP-binding domains, structural characteristics typical of ATP-binding cassette (ABC) proteins. Members of this superfamily, which include MDR proteins, are frequently involved in active transport of a variety of substrates across the cell membrane. Pulsed-field gel electrophoresis revealed the presence of 12 chromosomal bands in this clinical isolate of Cn. CneMDR1 was detected by hybridization on chromosome IV. High-stringency hybridization detected only one MDR-like gene. However, a second MDR-like gene (CneMDR2) was discovered during reverse transcriptase-PCR (RT-PCR) amplification using cDNA.

Identification and Characterization of a Monofunctional Glycosyltransferase from Staphylococcus aureus

A gene (mgt) encoding a monofunctional glycosyltransferase (MGT) from Staphylococcus aureus has been identified. This first reported gram-positive MGT shared significant homology with several MGTs from gram-negative bacteria and the N-terminal glycosyltransferase domain of class A high-molecular-mass penicillin-binding proteins from different species. S. aureus MGT contained an N-terminal hydrophobic domain perhaps involved with membrane association. It was expressed in Escherichia coli cells as a truncated protein lacking the hydrophobic domain and purified to homogeneity. Analysis by circular dichroism revealed that secondary structural elements of purified truncated S. aureus MGT were consistent with predicted structural elements, indicating that the protein might exhibit the expected folding. In addition, purified S. aureus MGT catalyzed incorporation of UDP-N-acetylglucosamine into peptidoglycan, proving that it was enzymatically active. MGT activity was inhibited by moenomycin A, and the reaction product was sensitive to lysozyme treatment. Moreover, a protein matching the calculated molecular weight of S. aureus MGT was identified from an S. aureus cell lysate using antibodies developed against purified MGT. Taken together, our results suggest that this enzyme is natively present in S. aureus cells and that it may play a role in bacterial cell wall biosynthesis.