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1.  Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli. 
Journal of Bacteriology  1993;175(20):6441-6450.
Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-beta-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake. Similar systems may be present in other gram-positive bacteria.
PMCID: PMC206752  PMID: 8407820
2.  Simultaneous detection of different Rhizobium strains marked with either the Escherichia coli gusA gene or the Pyrococcus furiosus celB gene. 
Applied and Environmental Microbiology  1996;62(11):4191-4194.
A new marker system for gram-negative bacteria was developed on the basis of the celB gene from the hyperthermophilic archaeon Pyrococcus furiosus, which encodes a thermostable beta-glucosidase with a high level of beta-galactosidase activity. The celB gene is highly suitable as a marker for studying plant-bacterium interaction because endogenous background beta-glucosidase and beta-galactosidase enzyme activity can readily be inactivated by heat and because inexpensive substrates for detection are commercially available. Two celB-expressing transposons were constructed for use in ecological studies of a variety of gram-negative bacteria. The combined use of the gusA marker gene and celB allowed the simultaneous detection of several Rhizobium strains on a plant, and multiple-strain occupancy of individual modules also could be easily detected.
PMCID: PMC168240  PMID: 8900009
3.  Transcriptional Regulation in the Hyperthermophilic Archaeon Pyrococcus furiosus: Coordinated Expression of Divergently Oriented Genes in Response to β-Linked Glucose Polymers 
Journal of Bacteriology  1999;181(12):3777-3783.
The genetic organization, expression, and regulation of the celB locus of the hyperthermophilic archaeon Pyrococcus furiosus were analyzed. This locus includes the celB gene, which codes for an intracellular β-glucosidase, and a divergently orientated gene cluster, adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular β-1,3-endoglucanase that is transcribed as a polycistronic messenger (the lamA operon). During growth of P. furiosus on either the β-1,4-linked glucose dimer cellobiose or the β-1,3-linked glucose polymer laminarin, the activities of both β-glucosidase and endoglucanase were increased at least fivefold compared with levels during growth on maltose or pyruvate. Northern blot analysis revealed an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon were identified 25 nucleotides downstream of conserved TATA box motifs. A number of repeating sequences have been recognized in the celB-adhA intergenic region, some of which might be part of a transcriptional regulator-binding site.
PMCID: PMC93856  PMID: 10368153
4.  Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli. 
Journal of Bacteriology  1995;177(24):7105-7111.
The celB gene encoding the cellobiose-hydrolyzing enzyme beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of beta-glucosidase that could be purified by a two-step purification procedure. The beta-glucosidase produced by E. coli had kinetic and stability properties similar to those of the beta-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of beta-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus beta-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200- or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100 degrees C.
PMCID: PMC177588  PMID: 8522516
5.  Correction of the beta-mannanase domain of the celC pseudogene from Caldocellulosiruptor saccharolyticus and activity of the gene product on kraft pulp. 
The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.
PMCID: PMC167498  PMID: 7793947
6.  Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells. 
Specific transfer and expression of bacterial luciferase genes via bacteriophages provides an efficient way to detect and assay viable host cells. Listeria bacteriophage A511 is a genus-specific, virulent myovirus which infects 95% of Listeria monocytogenes serovar 1/2 and 4 cells. We constructed recombinant derivative A511::luxAB, which carries the gene for a fused Vibrio harveyi LuxAB protein inserted immediately downstream of the major capsid protein gene (cps). Efficient transcription is initiated by the powerful cps promoter at 15 to 20 min postinfection. Site-specific introduction of the luciferase gene into the phage genome was achieved by homologous recombination in infected cells between a plasmid carrying A511 DNA flanking luxAB and phage DNA. Recombinants occurred in the lysate at a frequency of 5 x 10(-4) and were readily identified by the bioluminescent phenotype conferred on newly infected host cells. A511::luxAB can be used to directly detect Listeria cells. Following infection and a 2-h incubation period, numbers as low as 5 x 10(2) to 10(3) cells per ml were detected by using a single-tube luminometer. Extreme sensitivity was achieved by including an enrichment step prior to the lux phage assay; under these conditions less than 1 cell of L. monocytogenes Scott A per g of artificially contaminated salad was clearly identified. The assay is simple, rapid, inexpensive, and easy to perform. Our findings indicate that A511::luxAB is useful for routine screening of foods and environmental samples for Listeria cells.
PMCID: PMC167878  PMID: 8919773
7.  Bacteriophage biocontrol of Listeria monocytogenes on soft ripened white mold and red-smear cheeses 
Bacteriophage  2011;1(2):94-100.
Soft-ripened cheeses belong to the type of food most often contaminated with Listeria monocytogenes, and they have been implicated in several outbreaks of listeriosis. Bacteriophages represent an attractive way to combat foodborne pathogens without affecting other properties of the food. We used the broad host range, virulent Listeria phage A511 for control of L. monocytogenes during the production and ripening phases of both types of soft-ripened cheeses, white mold (Camembert-type) cheese, as well as washed-rind cheese with a red-smear surface (Limburger-type). The surfaces of young, unripened cheese were inoculated with 101–103 cfu/cm2 L. monocytogenes strains Scott A (serovar 4b) or CNL 103/2005 (serovar 1/2a). Phage was applied at defined time points thereafter, in single or repeated treatments, at 3 × 108 or 1 × 109 pfu/cm2. With Scott A (103 cfu/cm2) and a single dose of A511 (3 × 108 pfu/cm2) on camembert-type cheese, viable counts dropped 2.5 logs at the end of the 21 day ripening period. Repeated phage application did not further inhibit the bacteria, whereas a single higher dose (1 × 109 pfu/cm2) was found to be more effective. On red-smear cheese ripened for 22 days, Listeria counts were down by more than 3 logs. Repeated application of A511 further delayed re-growth of Listeria, but did not affect bacterial counts after 22 days. With lower initial Listeria contamination (101–102 cfu/cm2), viable counts dropped below the limit of detection, corresponding to more than 6 logs reduction compared to the control. Our data clearly demonstrate the potential of bacteriophage for biocontrol of L. monocytogenes in soft cheese.
doi:10.4161/bact.1.2.15662
PMCID: PMC3278646  PMID: 22334865
Listeria monocytogenes; bacteriophage; food safety; soft-ripened cheese
8.  Regulation of Endo-Acting Glycosyl Hydrolases in the Hyperthermophilic Bacterium Thermotoga maritima Grown on Glucan- and Mannan-Based Polysaccharides 
The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley β-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 109 cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting β-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative β-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular, β-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (Mr, 76,900) and Cel5A (Mr, 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99°C and an optimun temperature of 90°C, compared to 90 and 80°C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley β-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.
doi:10.1128/AEM.68.2.545-554.2002
PMCID: PMC126696  PMID: 11823189
9.  Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods. 
A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells. In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure. With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells. In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage. In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal. Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method. The standard plating procedure detected 57 positive samples. Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples. Overall, both methods performed similarly, i.e., were equally sensitive. However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method. Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described. The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora.
PMCID: PMC168593  PMID: 9251182
10.  Cellobiose-Specific Phosphotransferase System of Klebsiella pneumoniae and Its Importance in Biofilm Formation and Virulence 
Infection and Immunity  2012;80(7):2464-2472.
Klebsiella pneumoniae is a Gram-negative bacillus belonging to the family Enterobacteriaceae. In the past 20 years, K. pneumoniae has become the predominant pathogen causing community-acquired pyogenic liver abscess (PLA). The formation of biofilm facilitates bacterial colonization and has been implicated in reduced susceptibility to the host immune response. To investigate genes related to biofilm formation in a PLA-associated K. pneumoniae strain, a transposon mutant library was screened by microtiter plate assay to identify isolates impaired for biofilm formation. One of the mutants was disrupted in celB, encoding the putative cellobiose-specific subunit IIC of enzyme II (EIIC) of a carbohydrate phosphotransferase system (PTS). This transmembrane protein is responsible for recognizing and binding specific sugars and transporting them across the cell membrane into the cytoplasm. Deletion and chromosomal complementation of celB confirmed, by microtiter plate and slide culture assays, that celB was indeed responsible for biofilm formation. Cellobiose-specific PTS activities of deletion mutants grown in LB broth and 0.005% cellobiose minimal medium were markedly lower than that of the wild-type strain grown under the same conditions, thereby confirming the involvement of celB in cellobiose transport. In 0.005% cellobiose minimal medium, the celB mutant showed a delay in growth compared to the wild-type strain. In a mouse model of intragastric infection, deletion of the celB gene increased the survival rate from 12.5% to 87.5%, which suggests that the celB deletion mutant also exhibited reduced virulence. Thus, the celB locus of K. pneumoniae may contribute to biofilm formation and virulence through the metabolism of cellobiose.
doi:10.1128/IAI.06247-11
PMCID: PMC3416469  PMID: 22566508
11.  Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon. 
The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment.
PMCID: PMC168329  PMID: 9023917
12.  Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB from Thermotoga neapolitana† 
Applied and Environmental Microbiology  1998;64(12):4774-4781.
Two thermostable endocellulases, CelA and CelB, were purified from Thermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high Vmax value and low apparent Km value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside the Vmax values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding the celA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA and celB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.
PMCID: PMC90921  PMID: 9835561
13.  Identification of a Cellobiose Utilization Gene Cluster with Cryptic β-Galactosidase Activity in Vibrio fischeri▿  
Applied and Environmental Microbiology  2008;74(13):4059-4069.
Cellobiose utilization is a variable trait that is often used to differentiate members of the family Vibrionaceae. We investigated how Vibrio fischeri ES114 utilizes cellobiose and found a cluster of genes required for growth on this β-1,4-linked glucose disaccharide. This cluster includes genes annotated as a phosphotransferase system II (celA, celB, and celC), a glucokinase (celK), and a glucosidase (celG). Directly downstream of celCBGKA is celI, which encodes a LacI family regulator that represses cel transcription in the absence of cellobiose. When the celCBGKAI gene cluster was transferred to cellobiose-negative strains of Vibrio and Photobacterium, the cluster conferred the ability to utilize cellobiose. Genomic analyses of naturally cellobiose-positive Vibrio species revealed that V. salmonicida has a homolog of the celCBGKAI cluster, but V. vulnificus does not. Moreover, bioinformatic analyses revealed that CelG and CelK share the greatest homology with glucosidases and glucokinases in the phylum Firmicutes. These observations suggest that distinct genes for cellobiose utilization have been acquired by different lineages within the family Vibrionaceae. In addition, the loss of the celI regulator, but not the structural genes, attenuated the ability of V. fischeri to compete for colonization of its natural host, Euprymna scolopes, suggesting that repression of the cel gene cluster is important in this symbiosis. Finally, we show that the V. fischeri cellobioase (CelG) preferentially cleaves β-d-glucose linkages but also cleaves β-d-galactose-linked substrates such as 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal), a finding that has important implications for the use of lacZ as a marker or reporter gene in V. fischeri.
doi:10.1128/AEM.00190-08
PMCID: PMC2446528  PMID: 18487409
14.  Regulation of Major Cellulosomal Endoglucanases of Clostridium thermocellum Differs from That of a Prominent Cellulosomal Xylanase 
Journal of Bacteriology  2005;187(7):2261-2266.
The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 h−1, the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 h−1) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate—the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.
doi:10.1128/JB.187.7.2261-2266.2005
PMCID: PMC1065243  PMID: 15774868
15.  Design and Optimization of Short DNA Sequences That Can Be Used as 5′ Fusion Partners for High-Level Expression of Heterologous Genes in Escherichia coli 
Applied and Environmental Microbiology  2013;79(21):6655-6664.
The 5′ terminal nucleotide sequence of a gene is often a bottleneck in recombinant protein production. The ifn-α2bS gene is poorly expressed in Escherichia coli unless a translocation signal sequence (pelB) is fused to the 5′ end of the gene. A combined in silico and in vivo analysis reported here further indicates that the ifn-α2bS 5′ coding sequence is suboptimal for efficient gene expression. ifn-α2bS therefore presents a suitable model gene for describing properties of 5′ fusions promoting expression. We show that short DNA sequences corresponding to the 5′ end of the highly expressed celB gene, whose protein product is cytosolic, can functionally replace pelB as a 5′ fusion partner for efficient ifn-α2bS expression. celB fusions of various lengths (corresponding to a minimum of 8 codons) led to more than 7- and 60-fold stimulation of expression at the transcript and protein levels, respectively. Moreover, the presence of a celB-based fusion partner was found to moderately reduce the decay rate of the corresponding transcript. The 5′ fusions thus appear to act by enhancing translation, and bound ribosomes may accordingly contribute to increased mRNA stability and reduced mRNA decay. However, other effects, such as altered protein stability, cannot be excluded. We also developed an experimental protocol that enabled us to identify improved variants of the celB fusion, and one of these (celBD11) could be used to additionally increase ifn-α2bS expression more than 4-fold at the protein level. Interestingly, celBD11 also stimulated greater protein production of three other medically important human genes than the wild-type celB fragment.
doi:10.1128/AEM.01676-13
PMCID: PMC3811499  PMID: 23974137
16.  Bacteriophage amplification assay for detection of Listeria spp. using virucidal laser treatment 
Brazilian Journal of Microbiology  2012;43(3):1128-1136.
A protocol for the bacteriophage amplification technique was developed for quantitative detection of viable Listeria monocytogenes cells using the A511 listeriophage with plaque formation as the end-point assay. Laser and toluidine blue O (TBO) were employed as selective virucidal treatment for destruction of exogenous bacteriophage. Laser and TBO can bring a total reduction in titer phage (ca. 108 pfu/mL) without affecting the viability of L. monocytogenes cells. Artificially inoculated skimmed milk revealed mean populations of the bacteria as low as between 13 cfu/mL (1.11 log cfu/mL), after a 10-h assay duration. Virucidal laser treatment demonstrated better protection of Listeria cells than the other agents previously tested. The protocol was faster and easier to perform than standard procedures. This protocol constitutes an alternative for rapid, sensitive and quantitative detection of L. monocytogenes.
doi:10.1590/S1517-838220120003000040
PMCID: PMC3768892  PMID: 24031937
Listeria; bacteriophage A511; laser light; detection; skimmed milk
17.  Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-To-Eat Foods▿  
Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 × 103 CFU/g), phage added thereafter (3 × 106 to 3 × 108 PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 × 108 PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log10. In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.
doi:10.1128/AEM.01711-08
PMCID: PMC2612219  PMID: 19011076
18.  celB, a gene coding for a bifunctional cellulase from the extreme thermophile "Caldocellum saccharolyticum". 
Applied and Environmental Microbiology  1990;56(10):3117-3124.
"Caldocellum saccharolyticum" is an obligatory anaerobic thermophilic bacterium. A gene from this organism, designated celB, has been cloned in Escherichia coli as part of a bacteriophage lambda gene library. This gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose. The sequence of celB has homology with both exo- and endoglucanases described by others. It appears to have a central domain without enzymatic activity which is joined to the enzymatic domains by runs of amino acids rich in proline and threonine (PT boxes). Deletion analysis shows that the exoglucanase activity is located in the amino-terminal domain of the enzyme and that endoglucanase activity is located in the carboxy-terminal domain. There are internal transcriptional and translational start sites within the gene. The intact gene has been cloned into a temperature-inducible expression vector, pJLA602, and overexpressed in E. coli. Polyacrylamide gel electrophoresis showed that celB produced a protein with a molecular weight of 118,000 to 120,000. A number of smaller proteins with activity against carboxymethyl cellulose and 4-methyl umbelliferyl-beta-D-cellobioside were also produced. These are believed to be the result of alternative translational start sites and/or proteolytic degradation products of the translated gene product.
Images
PMCID: PMC184908  PMID: 2126700
19.  The Terminally Redundant, Nonpermuted Genome of Listeria Bacteriophage A511: a Model for the SPO1-Like Myoviruses of Gram-Positive Bacteria▿ † 
Journal of Bacteriology  2008;190(17):5753-5765.
Only little information on a particular class of myoviruses, the SPO1-like bacteriophages infecting low-G+C-content, gram-positive host bacteria (Firmicutes), is available. We present the genome analysis and molecular characterization of the large, virulent, broad-host-range Listeria phage A511. A511 contains a unit (informational) genome of 134,494 bp, encompassing 190 putative open reading frames (ORFs) and 16 tRNA genes, organized in a modular fashion common among the Caudovirales. Electron microscopy, enzymatic fragmentation analyses, and sequencing revealed that the A511 DNA molecule contains linear terminal repeats of a total of 3,125 bp, encompassing nine small putative ORFs. This particular genome structure explains why A511 is unable to perform general transduction. A511 features significant sequence homologies to Listeria phage P100 and other morphologically related phages infecting Firmicutes such as Staphylococcus phage K and Lactobacillus phage LP65. Equivalent but more-extensive terminal repeats also exist in phages P100 (∼6 kb) and K (∼20 kb). High-resolution electron microscopy revealed, for the first time, the presence of long tail fibers organized in a sixfold symmetry in these viruses. Mass spectrometry-based peptide fingerprinting permitted assignment of individual proteins to A511 structural components. On the basis of the data available for A511 and relatives, we propose that SPO1-like myoviruses are characterized by (i) their infection of gram-positive, low-G+C-content bacteria; (ii) a wide host range within the host bacterial genus and a strictly virulent lifestyle; (iii) similar morphology, sequence relatedness, and collinearity of the phage genome organization; and (iv) large double-stranded DNA genomes featuring nonpermuted terminal repeats of various sizes.
doi:10.1128/JB.00461-08
PMCID: PMC2519532  PMID: 18567664
20.  Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis 
Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa l-alanoyl-d-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-l-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SPslpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SPslpA–ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3′-end coding sequence of SPslpA–ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511Δ(S262–K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SPSlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511ΔC into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.
PMCID: PMC92096  PMID: 10877791
21.  Brochothrix thermosphacta Bacteriophages Feature Heterogeneous and Highly Mosaic Genomes and Utilize Unique Prophage Insertion Sites ▿ †  
Journal of Bacteriology  2010;192(20):5441-5453.
Brochothrix belongs to the low-GC branch of Gram-positive bacteria (Firmicutes), closely related to Listeria, Staphylococcus, Clostridium, and Bacillus. Brochothrix thermosphacta is a nonproteolytic food spoilage organism, adapted to growth in vacuum-packaged meats. We report the first genome sequences and characterization of Brochothrix bacteriophages. Phage A9 is a myovirus with an 89-nm capsid diameter and a 171-nm contractile tail; it belongs to the Spounavirinae subfamily and shares significant homologies with Listeria phage A511, Staphylococcus phage Twort, and others. The A9 unit genome is 127 kb long with 11-kb terminal redundancy; it encodes 198 proteins and 6 tRNAs. Phages BL3 and NF5 are temperate siphoviruses with a head diameter of 56 to 59 nm. The BL3 tail is 270 nm long, whereas NF5 features a short tail of only 94 nm. The NF5 genome (36.95 kb) encodes 57 gene products, BL3 (41.52 kb) encodes 65 products, and both are arranged in life cycle-specific modules. Surprisingly, BL3 and NF5 show little relatedness to Listeria phages but rather demonstrate relatedness to lactococcal phages. Peptide mass fingerprinting of viral proteins indicate programmed −1 translational frameshifts in the NF5 capsid and the BL3 major tail protein. Both NF5 and BL3 feature circularly permuted, terminally redundant genomes, packaged by a headful mechanism, and integrases of the serine (BL3) and tyrosine (NF5) types. They utilize unique target sequences not previously described: BL3 inserts into the 3′ end of a RNA methyltransferase, whereas NF5 integrates into the 5′-terminal part of a putative histidinol-phosphatase. Interestingly, both genes are reconstituted by phage sequence.
doi:10.1128/JB.00709-10
PMCID: PMC2950505  PMID: 20709901
22.  FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION 
1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]plaq., [extracellular phage]act., and [extracellular phage]plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück's objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.
PMCID: PMC2142814  PMID: 19873475
23.  Mechanism of cellulose synthesis in Agrobacterium tumefaciens. 
Journal of Bacteriology  1995;177(4):1076-1081.
Extracts of Agrobacterium tumefaciens incorporated UDP-[14C]glucose into cellulose. When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose. A combination of the membrane and soluble fractions restored the activity found in the original extracts. Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose. When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis. No synthesis was observed when extracts of mutants from the same group were mixed. The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A. G. Matthysse, S. White, and R. Lightfoot. J. Bacteriol. 177:1069-1075, 1995). Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose. When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed. In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-[14C]glucose into material extractable with organic solvents. No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon. The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants. The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis.
PMCID: PMC176704  PMID: 7860586
24.  Cellobiose-6-Phosphate Hydrolase (CelF) of Escherichia coli: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases 
Journal of Bacteriology  1999;181(23):7339-7345.
The gene celF of the cryptic cel operon of Escherichia coli has been cloned, and the encoded 6-phospho-β-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.86]) has been expressed and purified in a catalytically active state. Among phospho-β-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD+ for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-β-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-β-glucoside-6P, and the chromogenic analog, p-nitrophenyl-β-d-glucopyranoside-6P. In the absence of a metal ion and NAD+, purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD+ appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (Mw, ∼200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (Mr, ∼50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues; Mr = 50,512). Comparative sequence alignments provide tentative identification of the NAD+-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu112 and Glu356) of CelF.
PMCID: PMC103698  PMID: 10572139
25.  Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans. 
Journal of Bacteriology  1990;172(3):1576-1586.
Endo-beta-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subtilis PAP115 endo-beta-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, S1 nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ alpha-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum celB endoglucanase sequence but very little similarity to endoglucanases from other Bacillus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding.
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PMCID: PMC208635  PMID: 2307659

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