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PubMed1991

Highly conserved structure of proliferating cell nuclear antigen (DNA polymerase delta auxiliary protein) gene in plants.

In a previous paper [Suzuka, I., Daidoji, H., Matsuoka, M., Kadowaki, K., Takasaki, Y., Nakane, P. K. & Moriuchi, T. (1989) Proc. Natl Acad. Sci. USA 86, 3189-3193], we demonstrated the presence of a rice genomic-DNA fragment which hybridized with a rat proliferating-cell nuclear antigen (PCNA) cDNA probe, and proposed that plants possess the homolog of the PCNA gene which plays an essential role in DNA replication in mammals. Here we show that the sequence of the rice PCNA gene supports this proposal. The rice gene consists of four exons. The 5'-flanking region contains a variety of sequences similar to several consensus sequences believed to be important for the regulation of gene expression in eukaryotes. Determination of the transcription-start site and the structure of the 3'-flanking region of the gene indicated that the coded mRNA is 1073 bases. The rice PCNA genomic and cDNA clones are composed of 789 nucleotides; they encode a putative protein of 263 amino acids having a molecular mass of 29275 Da. Comparison of the coding regions of rice PCNA and rat PCNA revealed about 64% similarity at the nucleotide level and about 62% similarity at the amino acid level. Furthermore, comparison of the predicted amino acid sequence of the soybean PCNA with the rice PCNA sequence indicated a high degree of similarity (88%). The structural similarity between PCNA from plants and animals, despite the distant evolutionary relationship, indicates a strong selection pressure for conservation of this structure and suggests the presence of conserved DNA-replication mechanisms throughout the eukaryotes.

Amino Acid Sequence↗

PubMed1991

Structure of the human brain calcium-binding protein calretinin and its expression in bacteria.

Calbindin D28k and calretinin are two closely related intracellular calcium-binding proteins belonging to the troponin C superfamily. Calbindin is known to be involved in the vitamin-D-dependent calcium absorption through intestinal and renal epithelia, while the function of neuronal calbindin and calretinin is poorly understood. Using antibodies directed against chick intestinal calbindin D28k, human calretinin cDNA clones were isolated from brain cDNA libraries. The sequence of the calretinin cDNA revealed an open reading frame of 271 codons coding for a protein of 31,520 Da, and sharing 58% identical residues with human calbindin D28k. Calretinin contains five presumably active and one presumably inactive calcium-binding domains. Comparison with the partial sequences available for chick and guinea pig calretinins revealed that the protein is highly conserved in evolution (evolutionary rate: 0.27 x 10(-9) amino acid-1 year-1). The calretinin message was detected in the brain, while absent from heart muscle, kidney, liver, lung, spleen, stomach and thyroid gland. Recombinant calretinin was expressed in Escherichia coli, and the calcium-binding properties were confirmed on both the natural and the recombinant proteins. Part of the human gene coding for calretinin was isolated and the region corresponding to the promoter and the first exon was sequenced.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and expression of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase from mouse brain.

DNA clones encoding beta-galactoside alpha 2,3-sialyltransferase have been isolated from mouse brain cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open reading frame coding for 337 amino acids, and the deduced amino acid sequence showed 80% identity with that of porcine submaxillary gland Gal beta 1,3GalNAc alpha 2,3-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the NH2-terminal part including the cytoplasmic tail, signal-anchor domain and stem region was replaced with an immuno-globulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. This enzyme exhibited the transferase activity toward only the disaccharide moiety of Gal beta 1,3GalNAc of glycoproteins and glycolipids, no significant activity being detected for the other substrates tested.

Amino Acid Sequence↗

PubMed1994

Expression, assembly and secretion of a fully active plant ferredoxin-NADP+ reductase by Saccharomyces cerevisiae.

The flavoprotein ferredoxin-NADP+ reductase catalyzes the final step of the photosynthetic electron transport i.e., the reduction of NADP+ by ferredoxin. Expression and secretion of this enzyme was examined in Saccharomyces cerevisiae using a cDNA cloned from a pea library [Newman, B. J. & Gray, J. C. (1988) Plant Mol. Biol. 10, 511-520]. Two pea library cDNA sequences were employed, one corresponding to the mature enzyme and the other containing, in addition, the sequence of the transit peptide that directs ferredoxin-NADP+ reductase to the chloroplast. These sequences were introduced into a yeast shuttle vector in frame with the mating factor alpha 1 secretion-signal coding region under the control of its natural mating factor alpha 1 promoter. Saccharomyces cerevisiae cells transformed with the recombinant plasmids were able to synthesize and secrete fully active pea ferredoxin-NADP+ reductase. In both cases, a 35-kDa polypeptide was the major product. N-terminal sequencing of the secreted proteins indicates processing at position -1 with respect to the N-terminus of the pea mature enzyme. Yeast cells transformed with plasmid encoding the ferredoxin-NADP+ reductase precursor secrete four-times more ferredoxin-NADP+ reductase to the medium than cells transformed with the plasmid encoding the mature form of the enzyme. Ferredoxin-NADP+ reductases purified from culture medium showed structural and enzymatic properties that were identical, within the experimental error, to those of native plant ferredoxin-NADP+ reductase. The overall results indicate that pea ferredoxin-NADP+ reductase can be properly folded and its prosthetic group assembled in the yeast endoplasmic reticulum, and that its natural transit peptide favors its secretion.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and expression of chick embryo Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Comparison with the mammalian enzyme.

DNA clones encoding beta-galactoside alpha 2,6-sialyltransferase have been isolated from chick embryonic cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open-reading frame coding for 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region and a large C-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Gal beta 1,4GlcNAc in glycoproteins.

Amino Acid Sequence↗

PubMed2004

Characterization of a cathepsin L-associated protein in Artemia and its relationship to the FAS-I family of cell adhesion proteins.

We reported previously that the major cysteine protease in embryos and larvae of the brine shrimp, Artemia franciscana, is a heterodimeric protein consisting of a catalytic subunit (28.5 kDa) with a high degree of homology with cathepsin L, and a noncatalytic subunit (31.5 kDa) of unknown function. In the study reported here the noncatalytic subunit, or cathepsin L-associated protein (CLAP), was separated from cathepsin L by chromatography on Mono S and found to contain multiple isoforms with pIs ranging from 5.9 to 6.1. Heterodimeric and monomeric cathepsin L showed similar activity between pH 5 and 6.5, while the heterodimer was about twice as active as monomeric cathepsin L below pH 5. The heterodimer was more stable than the monomer between pH 6 and 7.4 and at 30-50 degrees C. Artemia CLAP and cathepsin L are present in nearly equimolar amounts at all stages in the life cycle and most abundant in encysted eggs and embyros. Moreover, CLAP, either free or as a complex with cathepsin L, was resistant to hydrolysis by cathepsin L. Two clones coding for CLAP were isolated from an Artemia embryo cDNA library and sequenced. Both clones have nearly identical open reading frames, but show differences at the 5'- and 3'-termini. Each cDNA clone has an extensive 3'-untranslated region containing 70-72% A+T. The deduced amino acid sequence of CLAP cDNA revealed two domains which were very similar to domains in fasciclin I and other cell adhesion proteins. The nucleotide sequences of clones 1 and 2 have been entered into the NCBI database (AY307377 and AY462276). This study supports the view that the noncatalytic subunit of the heterodimeric cysteine protease in Artemia stabilizes cathepsin L at various pH and temperatures normally inconsistent with cathepsin L from other organisms, and that CLAP serves as a docking mechanism for cathepsin L at nonlysosomal sites in Artemia embryos.

Amino Acid Sequence↗

PubMed2001

Structure and diversity of Gram-negative sulfate-reducing bacteria on rice roots.

Specific PCR assays were used to amplify the 16S rRNA genes of the Desulfobacteriaceae and the Desulfovibrionaceae from extracted environmental DNA from rice roots. 16S rDNA-based community patterns of the Desulfobacteriaceae were generated via terminal restriction fragment length polymorphism analysis from rice roots and compared with bulk soil. The molecular fingerprints showed no significant difference between rice roots and bulk soil, but changes during the vegetation period. 16S rDNA clone libraries and sequencing showed that the predominant terminal restriction fragments represented distinct phylogenetic groups. The 16S rDNA clone sequences of the Desulfobacteriaceae fell in the phylogenetic radiation of Desulfonema and Desulfosarcina or grouped within the Desulforhabdus-Syntrophobacter assemblage. Three of the latter sequences were closely affiliated with the MPN isolate EZ-2C2 from rice roots. All Desulfovibrionaceae 16S rDNA clone sequences, with one exception, were affiliated with the MPN isolate F1-7b from rice roots. The clustering of the clone sequences and the close phylogenetic affiliation with isolates from MPN enrichments from the same habitat in two cases indicated that these sequence clusters may represent predominant Gram-negative sulfate reducers on rice roots. Quantification of the bacterial abundances was accomplished by rRNA dot blot hybridization. In total the Gram-negative sulfate reducers accounted for approximately 2-3% of the total rRNA content. The relative rRNA abundance of the Desulfobacteriaceae was, at 1.4%, higher than that of the Desulfovibrionaceae (0.5%).

Journal Article↗

PubMed2004

Streptomyces coelicolor A3(2) lacks a genomic island present in the chromosome of Streptomyces lividans 66.

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage phiHAU3 resistance (phiHAU3r) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.

Base Sequence↗

PubMed2004

Characterization of the arginine deiminase operon of Streptococcus rattus FA-1.

The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the arc operon of Streptococcus rattus, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an arcB gene fragment obtained by degenerate PCRs, the FA-1 arc operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in S. rattus FA-1 are organized as an arcABCDT-adiR operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The arcA transcriptional start site was identified by primer extension, and a sigma(70)-like promoter was mapped 5' to arcA. Reverse transcriptase PCR was used to establish that arcABCDT could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.

Base Sequence↗

PubMed1994

The Vibrio cholerae toxin-coregulated-pilus gene tcpI encodes a homolog of methyl-accepting chemotaxis proteins.

Virulence gene activation in Vibrio cholerae is under the control of the ToxR-ToxT regulatory cascade. The ToxR regulon consists of genes required for toxin-coregulated-pilus (TCP) biogenesis, accessory colonization factor genes, cholera toxin genes, and ToxR-activated genes (tag) of unknown function. The tagB gene was isolated by using a tagB::TnphoA fusion junction to probe a V. cholerae )395 bacteriophage lambda library. Nucleotide sequence analysis revealed that tagB is identical to tcpI, a gene which encodes a protein that negatively regulates the synthesis of the major pilin subunit of TCP (TcpA). Our results show that the tcpI gene encodes a 620-amino-acid protein that shares extensive sequence similarity with the highly conserved signaling domain in methyl-accepting chemotaxis proteins. Expression of tcpI in Escherichia coli results in the synthesis of a 71-kDa polypeptide that becomes localized to the inner membrane. Similarly, TcpI-PhoA alkaline phosphatase activity is enriched in V. cholerae inner membrane preparations. Colonies of V. cholerae tcpI::TnphoA mutant cells display increased swarming on solid media when compared with those of the parental V. cholerae O395. Taken together, these observations suggest that TcpI may play a dual role in promoting vibrio colonization of the small bowel. In response to the appropriate environmental signal(s), TcpI permits maximum expression of tcpA while simultaneously reducing vibrio chemotaxis-directed motility. We believe coordinate regulation of colonization and motility determinants, in such a fashion, facilitates efficient V. cholerae microcolony formation.

Amino Acid Sequence↗

PubMed1994

The Vibrio cholerae acfB colonization determinant encodes an inner membrane protein that is related to a family of signal-transducing proteins.

Vibrio cholerae accessory colonization factor genes (acfA, B, C, and D) are required for efficient intestinal colonization. Expression of acf genes is under the control of a regulatory cascade that also directs the synthesis of cholera toxin and proteins involved in the biogenesis of the toxin-coregulated pilus. The gene for acfB was cloned by using an acfB::TnphoA fusion junction to probe a V. cholerae O395 bacteriophage lambda library. DNA sequence analysis revealed that acfB is predicted to encode a 626-amino-acid protein related to the V. cholerae HlyB and TcpI proteins. These three Vibrio proteins have amino acid sequence similarity in a region highly conserved among bacterial methyl-accepting chemotaxis proteins. Analysis of the predicted AcfB amino acid sequence suggests that this colonization determinant possesses a membrane topology and domain organization similar to those of methyl-accepting chemotaxis proteins. Heterologous expression of acfB in Escherichia coli generates four polypeptide species with apparent molecular masses of 34, 35, 74, and 75 kDa. The 74- and 75-kDa proteins appear to represent modified forms of the full-length AcfB protein. The 34- and 35-kDa polypeptide species most likely correspond to a C-terminal 274-amino-acid polypeptide that results from internal translation initiation of acfB mRNA. Localization studies with AcfB-PhoA hybrid proteins indicate that AcfB resides in the V. cholerae inner membrane. V. cholerae acfB::TnphoA mutants display an altered motility phenotype in semisolid agar. The relationship between AcfB and Vibrio motility and the amino acid similarities between AcfB and chemotaxis signal-transducing proteins suggest that AcfB may interact with the V. cholerae chemotaxis machinery. The data presented in this report provide preliminary evidence that acfB encodes an environmental sensor/signal-transducing protein involved in V. cholerae colonization.

Amino Acid Sequence↗

PubMed2000

Identification and expression of a Mycoplasma gallisepticum surface antigen recognized by a monoclonal antibody capable of inhibiting both growth and metabolism.

In order to identify antigenic proteins of Mycoplasma gallisepticum, monoclonal antibodies (MAbs) against virulent M. gallisepticum R strain were produced in mice. MAb 35A6 was selected for its abilities to inhibit both growth and metabolism of M. gallisepticum in vitro. The MAb recognized a membrane protein with an apparent molecular mass of 120 kDa. The corresponding gene, designated the mgc3 gene, was cloned from an M. gallisepticum genomic DNA expression library and sequenced. The mgc3 gene is a homologue of the ORF6 gene encoding 130-kDa protein in the P1 operon of M. pneumoniae and is localized downstream of the mgc1 gene, a homologue of the P1 gene. To assess the characteristics of MGC3 protein, all 10 TGA codons in the mgc3 gene, which encode a tryptophan in the Mycoplasma species, were replaced with TGG codons, and recombinant fowlpox viruses (FPV) harboring the altered mgc3 gene were constructed. One of the recombinant FPVs was improved to express MGC3 protein on the cell surface in which the signal peptide of MGC3 protein was replaced with one from Marek's disease virus gB. These results should provide the impetus to develop a vaccine based on MGC3 protein which can induce antibodies with both growth inhibition and metabolic-inhibition activities using a recombinant FPV.

Amino Acid Sequence↗

PubMed2002

Molecular analysis of group B protective surface protein, a new cell surface protective antigen of group B streptococci.

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and characterization of the Candida albicans enolase gene.

A DNA clone containing the putative Candida albicans enolase gene (ENO1) was isolated from a genomic DNA library. The sequenced insert contained a continuous open reading frame of 1,320 bp. The predicted 440-amino-acid protein is 78 and 76% identical, respectively, to Saccharomyces cerevisiae enolase proteins 1 and 2. Only one enolase gene could be detected in C. albicans genomic DNA by Southern analysis with a homologous probe. Northern (RNA) analysis detected a single, abundant C. albicans ENO1 transcript of approximately 1,600 nucleotides. When cells were grown on glucose, levels of ENO1 mRNA were markedly increased by comparison with ENO1 mRNA levels in cells grown on ethanol, a gluconeogenic carbon source. In contrast to this glucose-mediated transcriptional induction, the carbon source had no dramatic effect on the levels of enolase protein or enzyme activity in the C. albicans strains tested. These results suggest that posttranscriptional mechanisms are responsible for modulating expression of the C. albicans enolase gene.

Amino Acid Sequence↗

PubMed1997

Use of rpsL for dominance selection and gene replacement in Streptomyces roseosporus.

We developed a gene replacement system using the rpsL gene of Streptomyces roseosporus and demonstrated its utility by constructing a deletion in the S. roseosporus glnA gene. A 1.3-kb BamHI fragment that hybridized to the Mycobacterium smegmatis rpsL gene was subcloned from an S. roseosporus cosmid library and sequenced. Plasmid pRHB514 containing the rpsL gene conferred streptomycin sensitivity (Sm(S)) to the Sm(r) S. roseosporus TH149. The temperature-sensitive plasmid pRHB543 containing rpsL and the S. roseosporus glnA gene disrupted with a hygromycin resistance (Hm(r)) gene was introduced into S. roseosporus TH149, and recombinants containing single and double crossovers were obtained after a temperature increase. Southern hybridization analysis revealed that single crossovers occurred in the glnA or rpsL genes and that double crossovers resulted in replacement of the chromosomal glnA gene with the disrupted glnA. Glutamine synthetase activity was undetectable in the recombinant containing the disrupted glnA gene.

Amino Acid Sequence↗

PubMed1999

Cloning and expression of Mycobacterium tuberculosis and Mycobacterium leprae dihydropteroate synthase in Escherichia coli.

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

Cloning, Molecular↗

PubMed1993

Cloning of a DNA sequence unique to Clostridium botulinum group I by selective hybridization.

Nucleic acid sequences were isolated from a strain of Clostridium botulinum type A by a selective hybridization method known as deletion enrichment. Nontoxigenic C. sporogenes was used to produce a C. botulinum type A sequence-enriched library. A probe, pCBM44, which showed specific hybridization to a 4.0-kb HindIII fragment present in all of the C. botulinum type A strains tested was isolated, and there was no hybridization to any strains of C. sporogenes. Upon further investigation, pCBM44 was found to hybridize to all of the group I proteolytic C. botulinum strains tested (toxin types A, B, and F) but not to hybridize to groups II, III, and IV (toxin types B, C, D, or E). The probe did not cross-react with nine other Clostridium spp. Such a probe, which differentiates between nontoxigenic C. sporogenes and neurotoxigenic C. botulinum group I strains, should prove extremely useful.

Botulinum Toxins↗

PubMed2003

Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes.

Epidemiological studies and analysis of putative virulence genes have shown that Listeria monocytogenes has diverged into several phylogenetic divisions. We hypothesize that similar divergence has occurred for many genes that influence niche-specific fitness and virulence and that identifying these differences may offer new opportunities for the detection, treatment, and control of this important pathogen. To explore this issue further, we developed a microarray composed of fragmented DNA taken from 10 strains of L. monocytogenes. We then hybridized genomic DNA from 50 different strains to replicate arrays and analyzed the resulting hybridization patterns. A simple Euclidean distance metric permitted the reconstruction of previously described genetic relationships between serotypes, and only four microarray probes were needed to discriminate between the most important serotypes (1/2a, 1/2b, 1/2c, and 4). We calculated an index of linkage equilibrium from the microarray data and confirmed that L. monocytogenes has a strongly clonal population structure (I(A) = 3.85). Twenty-nine informative probes were retrieved from the library and sequenced. These included genes associated with repairing UV-damaged DNA, salt tolerance, biofilm formation, heavy metal transport, ferrous iron transport, and teichoic acid synthesis. Several membrane-bound lipoproteins and one internalin were identified, plus three phage sequences and six sequences with unknown function. Collectively, these data confirm that many genes have diverged between lineages of L. monocytogenes. Furthermore, these results demonstrate the value of mixed-genome microarrays as a tool for deriving biologically useful information and for identifying and screening genetic markers for clinically important microbes.

Cluster Analysis↗