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A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus. Juan G. Arrieta, 2004.The endophytic diazotroph Gluconacetobacter diazotrophicus secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10) to utilize plant sucrose . LsdA, unlike other extracellular levansucrases from gram-negative bacteria, is transported to the periplasm by a signal-peptide-dependent pathway . We identified an unusually organized gene cluster encoding at least the components LsdG, -O, -E, -F, -H, -I, -J, -L, -M, -N, and -D of a type II secretory system required for LsdA translocation across the outer membrane . Another open reading frame, designated lsdX, is located between the operon promoter and lsdG, but it was not identified in BLASTX searches of the DDBJ/EMBL/GenBank databases . The lsdX, -G, and -O genes were isolated from a cosmid library of strain SRT4 by complementation of an ethyl methanesulfonate mutant unable to transport LsdA across the outer membrane . The downstream genes lsdE, -F, -H, -I, -J, -L, -M, -N, and -D were isolated through chromosomal walking . The high G+C content (64 to 74%) and the codon usage of the genes identified are consistent with the G+C content and codon usage of the standard G . diazotrophicus structural gene . Sequence analysis of the gene cluster indicated that a polycistronic transcript is synthesized . Targeted disruption of lsdG, lsdO, or lsdF blocked LsdA secretion, and the bacterium failed to grow on sucrose . Replacement of Cys162 by Gly at the C terminus of the pseudopilin LsdG abolished the protein functionality, suggesting that there is a relationship with type IV pilins . Restriction fragment length polymorphism analysis revealed conservation of the type II secretion operon downstream of the levansucrase-levanase (lsdA-lsdB) locus in 14 G . diazotrophicus strains representing 11 genotypes recovered from four different host plants in diverse geographical regions . To our knowledge, this is the first report of a type II pathway for protein secretion in the Acetobacteraceae . Proteolytic Activity of YibP Protein in Escherichia coli. Toshiharu Ichimura, 2002.Escherichia coli YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family . A yibP disrupted mutant grows in rich medium at 37°C but not at 42°C . In the yibP null mutant, cell division and FtsZ ring formation are inhibited at 42°C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis . Five percent betaine suppresses the temperature sensitivity of the yibP disrupted mutation . The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain . YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction . Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space . Purified YibP has a protease activity that split the substrate ß-casein . Conserved Serine/Threonine Kinase Encoded by CBK1 Regulates Expression of Several Hypha-Associated Transcripts and Genes Encoding Cell Wall Proteins in Candida albicans. Mark D. McNemar, 2002.The opportunistic fungal pathogen, Candida albicans, is reported to have several potential virulence factors . A potentially significant factor is the ability to undergo morphological transition from yeast to hypha . This alteration of form is accompanied by many changes within the cell, including alterations in gene expression and cell wall composition . We have isolated a gene that encodes a highly conserved serine/threonine kinase that appears to be involved in the regulation of proteins associated with the cell wall . We have assigned the designation CBK1 (cell wall biosynthesis kinase 1) to this gene . Mutants lacking CBK1 form large aggregates of round cells under all growth conditions and lack the ability to undergo morphological differentiation . Additionally, these mutants show an altered pattern of expression of several transcripts encoding proteins associated with the cell wall . The results suggest that the kinase encoded by CBK1 plays a general role in the maintenance and alteration of the cell wall of C . albicans in all morphologies . Molecular Epidemiology of Mycoplasma conjunctivae in Caprinae: Transmission across Species in Natural Outbreaks. Luc Belloy, 2003.Mycoplasma conjunctivae is the etiological agent of infectious keratoconjunctivitis, a highly contagious ocular infection that affects both domestic and wild Caprinae species in the European Alps . In order to study the transmission and spread of M . conjunctivae across domestic and wild Caprinae populations, we developed a molecular method for subtyping and identifying strains of M . conjunctivae . This method is based on DNA sequence determination of a variable domain within the gene lppS, a gene that encodes an antigenic lipoprotein of M . conjunctivae . This domain of lppS shows variations among different strains but remains constant upon generations of individual strains on growth medium and thus allows identification of individual strains and estimation of their phylogenetic intercorrelations . The variable domain of lppS is amplified by PCR using primers that match conserved sequences of lppS flanking it . Sequence analysis of the amplified fragment enables fine subtyping of M . conjunctivae strains . The method is applicable both to isolated strains and to clinical samples directly without requiring the cultivation of the strain . Using this method, we show that M . conjunctivae was transmitted between domestic and wild animals that were grazing in proximate pastures . Certain animals also presented infections with two different strains simultaneously .
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