Lactose and D-galactose metabolism in Staphylococcus aureus. III. Purification and properties of D-tagatose-6-phosphate kinase

D-Tagatose-6-phosphate kinase, an inducible enzyme that functions in the metabolism of lactose and D-galactose in Staphylococcus aureus, was purified about 300-fold from an extract of D-galactose-grown cells. The enzyme catalyzed the nucleoside triphosphate-dependent phosphorylation of both D-tagatose 6-phosphate and D-fructose 6-phosphate. Although the Vmax values were equal for these two substrates, the apparent Km values differed by 10,000-fold, being 16 micro M for D-tagatose 6-phosphate and 150 mM for D-fructose 6-phosphate. The purified enzyme was free from the constitutive D-fructose-6-phosphate kinase. Phosphoryl donors used by D-tagatose-6-phosphate kinse, listed in order of decreasing rates at saturating concentrations were GTP, UTP ITP ATP, CTP, and TTP; the Km values were 0.38, 0.91, 0.17, 0.16, 18, and 20 mM, respectively. The enzyme appeared to be nonallosteric; it exhibited Michaelis-Menten kinetics and was not inhibited by high concentrations of MgATP. However, it was activated 3- to 4-fold by 33.3 mM K+, NH4+, Rb+, and Cs+, and was inhibited 31 to 65% by 33.3 mM Na+ and Li+. It was inactivated reversibly by the thiol reagent, N-ethylmaleimide. The subunit molecular weight was estimated to be 52,000, and the native enzyme appeared to be a dimer with a sedimentation coefficient of 6.8 S. Data on stability, pH optimum, and inducibility of the enzyme are also presented.     

Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization

It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research.     

IGS Minisatellites Useful for Race Differentiation in Colletotrichum lentis and a Likely Site of Small RNA Synthesis Affecting Pathogenicity

Colletotrichum lentis is a fungal pathogen of lentil in Canada but rarely reported elsewhere. Two races, Ct0 and Ct1, have been identified using differential lines. Our objective was to develop a PCR-probe differentiating these races. Sequences of the translation elongation factor 1α (tef1α), RNA polymerase II subunit B2 (rpb2), ATP citrate lyase subunit A (acla), and internal transcribed spacer (ITS) regions were monomorphic, while the intergenic spacer (IGS) region showed length polymorphisms at two minisatellites of 23 and 39 nucleotides (nt). A PCR-probe (39F/R) amplifying the 39 nt minisatellite was developed which subsequently revealed 1–5 minisatellites with 1–12 repeats in C. lentis. The probe differentiated race Ct1 isolates having 7, 9 or 7+9 repeats from race Ct0 having primarily 2 or 4 repeats, occasionally 5, 6, or 8, but never 7 or 9 repeats. These isolates were collected between 1991 and 1999. In a 2012 survey isolates with 2 and 4 repeats increased from 34% to 67%, while isolated with 7 or 9 repeats decreased from 40 to 4%, likely because Ct1 resistant lentil varieties had been grown. The 39 nt repeat was identified in C. gloeosporioides, C. trifolii, Ascochyta lentis, Sclerotinia sclerotiorum and Botrytis cinerea. Thus, the 39F/R PCR probe is not species specific, but can differentiate isolates based on repeat number. The 23 nt minisatellite in C. lentis exists as three length variants with ten sequence variations differentiating race Ct0 having 14 or 19 repeats from race Ct1 having 17 repeats, except for one isolate. RNA-translation of 23 nt repeats forms hairpins and has the appropriate length to suggest that IGS could be a site of small RNA synthesis, a hypothesis that warrants further investigation. Small RNA from fungal plant pathogens able to silence genes either in the host or pathogen thereby aiding infection have been reported.     

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.     

Diversity of root-endophytic <Emphasis Type="Italic">Trichoderma</Emphasis> from Malaysian Borneo

Trichoderma species form endophytic associations with plant roots and may provide a range of benefits to their hosts. However, few studies have systematically examined the diversity of Trichoderma species associated with plant roots in tropical regions. During the evaluation of Trichoderma isolates for use as biocontrol agents, root samples were collected from more than 58 genera in 35 plant families from a range of habitats in Malaysian Borneo. Trichoderma species were isolated from surface-sterilised roots and identified following analysis of partial translation elongation factor-1α (tef1) sequences. Species present included Trichoderma afroharzianum, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma guizhouense, Trichoderma reesei, Trichoderma strigosum and Trichoderma virens. Trichoderma asperellum/T. asperelloides, Trichoderma harzianum s.l. and T. virens were the most frequently isolated taxa. tef1 sequence data supported the recognition of undescribed species related to the T. harzianum complex. The results suggest that tropical plants may be a useful source of novel root-associated Trichoderma for biotechnological applications.     

C/N Ratio Drives Soil Actinobacterial Cellobiohydrolase Gene Diversity

Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle.     

Bacterial diversity in organically-enriched fish farm sediments

The bacterial diversity and community structure within both organically enriched and adjacent, unimpacted, near-shore marine sediments at two fish farms in southern Tasmania, Australia, was examined using 16S rRNA gene clone library construction and analysis. Sediments at both caged and reference sites at both farms showed a very high level of microbial diversity. Over 900 clones were analysed and grouped into 631 unique phylotypes. Reference sites were dominated by Delta- and Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cage site sediments were also dominated by these phylotypes, as well as members of the Alpha- and Epsilonproteobacteria. Diversity and coverage indices indicated that the actual diversity of the sediments was much greater than that detected, despite a large sampling effort. All libraries were shown to be statistically different from one another (P < 0.05). Many phylotypes did not group with cultured bacteria, but grouped with other environmental clones from a wide array of marine benthic environments. Diversity and evenness indices suggested that although both parameters changed after farming, diverse communities were present in all sediments. The response of the microbial community to organic load suggested that random, rather than predictable, succession events determine community composition and diversity, and that sediment type may influence bacterial community and sediment response to organic perturbation.     

OPTICAL MONITORING AND FORECASTING SYSTEMS FOR HARMFUL ALGAL BLOOMS: POSSIBILITY OR PIPE DREAM?

Monitoring programs for harmful algal blooms (HABs) are currently reactive and provide little or no means for advance warning. Given this, the development of algal forecasting systems would be of great use because they could guide traditional monitoring programs and provide a proactive means for responding to HABs. Forecasting systems will require near real-time observational capabilities and hydrodynamic/biological models designed to run in the forecast mode. These observational networks must detect and forecast over ecologically relevant spatial/ temporal scales. One solution is to incorporate a multiplatform optical approach utilizing remote sensing and in situ moored technologies. Recent advances in instrumentation and data-assimilative modeling may provide the components necessary for building an algal forecasting system. This review will outline the utility and hurdles of optical approaches in HAB detection and monitoring. In all the approaches, the desired HAB information must be isolated and extracted from the measured bulk optical signals. Examples of strengths and weaknesses of the current approaches to deconvolve the bulk optical properties are illustrated. After the phytoplankton signal has been isolated, species-recognition algorithms will be required, and we demonstrate one approach developed for Gymnodinium breve Davis. Pattern-recognition algorithms will be species-specific, reflecting the acclimation state of the HAB species of interest.Field data will provide inputs to optically based ecosystem models, which are fused to the observational networks through data-assimilation methods. Potential model structure and data-assimilation methods are reviewed.     

Injection tube differentiation in gun cells of a haptoglossa species which infects nematodes

The gun cells which develop from germinating cysts in Haptoglossa produce a specialized infection apparatus, the injection tube. Upon eversion this tube fires a missile-like projectile which penetrates the host cuticle and then forms an infective sporidium within the body cavity of the nematode host. The temporal assembly of this complex cell organelle has been determined by serial-section reconstructions of maturing gun cells in a previously undescribed Haptoglossa species. The differentiation of the partially walled inverted injection tube is an unusual example of internal tube growth, in which membrane and wall assembly are temporally separated. There is no evidence that the shape of this inverted tube, which coils around the nucleus until it doubles back on itself, is dictated by the disposition of cytoplasmic microtubules. However, actin-like material was associated with the delimiting membrane of the differentiating tube, particularly in the regions of extension. From these studies it seems likely that the "head and buttress" structures previously depicted as the barbed tip of the "harpoon-like" penetration missile are part of a separate, structurally complex system which we suggest locks the "missile" into position in the invaginated injection tube. From this detailed account of cell architecture, models for the likely mechanism of infection cell firing are discussed, and unresolved questions relating to the cell biology and biochemistry of these complex organelles are highlighted. Copyright 1998 Academic Press.