Publications

• Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides.

  Lai X., Davis F.C., Hespell R.B., Ingram L.O.

  Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibri ... >> More

  Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation. << Less

  Appl. Environ. Microbiol. 63:355-363(1997) [PubMed] [EuropePMC]

• Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli.

  Lai X., Ingram L.O.

  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 ... >> More

  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. << Less

  J. Bacteriol. 175:6441-6450(1993) [PubMed] [EuropePMC]

• The major PEP-phosphotransferase systems (PTSs) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth.

  Stoll R., Goebel W.

  In this report we examine the PEP-dependent phosphotransferase systems (PTSs) of Listeria monocytogenes EGD-e, especially those involved in glucose and cellobiose transport. This L. monocytogenes strain possesses in total 86 pts genes, encoding 29 complete PTSs for the transport of carbohydrates a ... >> More

  In this report we examine the PEP-dependent phosphotransferase systems (PTSs) of Listeria monocytogenes EGD-e, especially those involved in glucose and cellobiose transport. This L. monocytogenes strain possesses in total 86 pts genes, encoding 29 complete PTSs for the transport of carbohydrates and sugar alcohols, and several single PTS components, possibly supporting transport of these compounds. By a systematic deletion analysis we identified the major PTSs involved in glucose, mannose and cellobiose transport, when L. monocytogenes grows in a defined minimal medium in the presence of these carbohydrates. Whereas all four PTS permeases belonging to the PTS(Man) family may be involved in mannose transport, only two of these (PTS(Man)-2 and PTS(Man)-3), and in addition at least one (PTS(Glc)-1) of the five PTS permeases belonging to the PTS(Glc) family, are able to transport glucose, albeit with different efficiencies. Cellobiose is transported mainly by one (PTS(Lac)-4) of the six members belonging to the PTS(Lac) family. In addition, PTS(Glc)-1 appears to be also able to transport cellobiose. The transcription of the operons encoding PTS(Man)-2 and PTS(Lac)-4 (but not that of the operon for PTS(Man)-3) is regulated by LevR-homologous PTS regulation domain (PRD) activators. Whereas the growth rate of the mutant lacking PTS(Man)-2, PTS(Man)-3 and PTS(Glc)-1 is drastically reduced (compared with the wild-type strain) in the presence of glucose, and that of the mutant lacking PTS(Lac)-4 and PTS(Glc)-1 in the presence of cellobiose, replication of both mutants within epithelial cells or macrophages is as efficient as that of the wild-type strain. << Less

  Microbiology (Reading) 156:1069-1083(2010) [PubMed] [EuropePMC]

• Cellobiose-specific phosphotransferase system of Klebsiella pneumoniae and its importance in biofilm formation and virulence.

  Wu M.C., Chen Y.C., Lin T.L., Hsieh P.F., Wang J.T.

  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 im ... >> More

  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. << Less

  Infect Immun 80:2464-2472(2012) [PubMed] [EuropePMC]