The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues

Type IV pre-pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pili. The type IV pre-pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Acc l fragment on account of its hybridization with the DNA fragment containing the type IV pre-pilin leader-peptidase gene pilD/xcpA of Pseudomonas aeruginosa . Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO ) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre-pilin leader peptidases, XpsO is unique in not having the two conserved -CXXC- motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein-secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa , both the sensitivity against infection with the pilus-specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.     

Salmonella typhimurium secreted invasion determinants are homologous to Shigella lpa proteins

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes ( sspB , sspC , sspD , and sspA ), located between spaT and prgH , which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins lpaB, lpaC, lpaD and lpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87,42 and 36 kDa. Complementation analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA . sspC and sspD , but not sspA are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK , homologous to the Shigella lpa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63,59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri lpa proteins.     

Quelling the red menace: haem capture by bacteria

Haem is an important bacterial nutrient. As a prosthetic group of several proteins, haem functions as a cofactor mediating oxygen transport, energy generation, and mixed-function oxidation. In addition, the iron chelated in the porphyrin ring may serve as an iron substrate for growth. However, because of its propensity for oxidizing cellular constituents, haem is always associated with proteins. Therefore, the uptake and transit of haem across bacterial membranes requires the participation of protein escorts. Bacteria have evolved a diverse array of surface-exposed receptors dedicated to binding haem and haem-proteins. Following this selective recognition at the bacterial cell surface, haem is transported across the outer membrane via a TonB-dependent process. The control of receptor expression appears to be multifactorial, probably involving a number of global regulators. A model integrating this information is presented.     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.     

Screening and characterization of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp.

Summary Three screening methods were used to isolate GL-7-ACA acylase-producing strains. Three positive isolates were identified with Pseudomonas nitroreducens CCRC 11041 possessing the highest activity, against GL-7-ACA and GL-7-ADCA. No activity was detected when Ceph C or succinyl-7-ACA was used as substrate; glutaric acid was found to be inhibitory. CCRC 11041 could produce maximal GL-7-ACA acylase activity when cultivated on meat extract medium II. The enzyme had a pH optimum of 5.0 and a temperature optimum of 42°C.     

Distribution of toxigenic Bacillus cereus in rice samples marketed in Hong Kong

Eleven of 16 samples of rice on sale in rice shops and supermarkets in Hong Kong contained Bacillus cereus. Although B. cereus counts did not exceed 100 bacteria/g in most of the positive samples, a sample of Thai red rice and a poor quality rice originating from China contained between 300 to 1000 cells/g and 10⁴ to 2×10⁵ cells/g, respectively. Nine strains produced an enterotoxin responsible for the diarrhoeal-type B. cereus food poisoning and seven of these strains also produced a haemolysin (haemolysin BL), a dermonecrotic vascular permeability factor which may be a virulence determinant in diarrhoeal illness caused by this bacterium.P.K. Lee and J.A. Buswell are with the Department of Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong; K. Shinagawa is with the Department of Veterinary Medicine, Iwate University, Iwate, Japan.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

双碳源单细胞蛋白间歇培养及流加培养的动力学模型

应用非结构的逻辑增殖模型研究了两种酵母的单碳源和双碳源单细胞蛋白间歇培养的动力学,用改进的逻辑增殖模型研究了双碳源流加培养过程的动力学,从实验数据拟合了动力学模型参数,模型计算值与实验数据吻合良好。     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.