Location and characterization of two functions on RP1 that inhibit the fertility of the IncW plasmid R388

Two fertility-inhibition functions which reduce R388 (IncW) transfer were detected on RP1 (60 kb, IncP). The respective genes, fiwA and fiwB, were mapped by transposon insertion mutagenesis to the regions between coordinates 32.8 to 31.7 kb (fiwA), and 59.8 to 0.8 kb (fiwB). The fiwA function occurs in a non-essential region of RP1 whereas fiwB is straddled by essential plasmid-maintenance and host-range determinants and apparently coincides (or overlaps) with the gene for tellurite-resistance.     

The Surface Exclusion System of RP1: Investigation of the Roles of trbJ and trbK in the Surface Exclusion, Transfer, and Slow-Growth Phenotypes

The contiguous trbJ and trbK genes of RP1 were cloned individually to study their effects. Surface exclusion was conferred only by trbK and only when gene dosage was high or when trbJ was also present in cis or in trans. This suggests that in the low-copy-number RP1, surface exclusion is due to a two-gene interaction in which trbK is the dominant partner. Among surface exclusion genes, trbJ is novel in yielding a periplasmic product that is also essential for conjugal transfer. This cellular location and the disturbed membrane function that accompanies TrbJ-processing probably accounts for the retarded growth caused by trbJ⁺ clones in Pseudomonas aeruginosa strain PAO.     

Isolation and characterisation of deletion mutants involving the transfer genes of P-group plasmids in Pseudomonas aeruginosa

Summary The P-group plasmids RP1 and R26 are recovered at low frequency following conjugal transfer to B3-lysogens of P. aeruginosa PAO. The rare carbenicillin-resistant transcipients that do arise are usually transfer-defective (Tra^-) and may show the loss of other plasmid borne functions, namely kanamycin-resistance (Km^r) and reduced plating of phage GlOl (Spp⁺). The four phenotypic classes that occur among the Tra^- derivatives are respectively, Tra^- (69–81%), Tra^- Spp^- (12–30%), Tra^- Km^s and Tra^- Km^s Spp^- (0.2–1%), of which the latter three are due to plasmid deletions. This is seen from the sizes of the plasmids carried by these bacteria and from the transductional analysis of the R26-derivatives. Thus, although R26 (MW=52×10⁶ daltons) is too large to be transduced by phage F116L (MW=40×10⁶), this is possible for its Tra^- Km^s and Tra^- Km^s Spp^- derivatives. The phenotypes and frequencies of the various transcipient classes suggests that the gene order Km..Tra..Spp occurs in both RP1 and R26, and that Spp is more closely linked to Tra than is Km. These conclusions are supported by the sizes of the plasmid mutants since deletions spanning the loci Km Tra Spp, Km Tra, and Tra Spp involve the loss of DNA of MW 8-17×10⁶, 5-13×10⁶ and 1-9×10⁶ daltons respectively.Whilst all the transcipients displayed the incompatibility properties of the parent plasmids (Inc⁺), only some retained plasmid surface exclusion (Sfx⁺). Moreover, a strict correlation existed between the Sfx and Spp phenotypes such that the transcipients were either wild type, Sfx^- Spp^-, or displayed an intermediate phenotype for both characters. This suggests that these phenotypes are controlled by closely linked genes or are different manifestations of the same gene function. The deletion map of these various markers in both RP1 and R26 therefore seems to be Km..Tra..Sfx/Spp..Inc.     

Effect of Lincocin Treatment on the Greening Process in Bean (Phaseolus vulgaris) Leaves

The effect of lincocin (a plastid protein synthesis inhibitor) treatment on the greening process of bean (Phaseolus vulgaris L.) leaves have been studied. In comparison with control leaves treated ones had a decreased rate of chloroplast development. They had a marked chlorophyll deficiency and a decreased chlorophyll a/b ratio. Some long and short wavelength forms of chlorophyll a were lacking as evidenced from the absorption spectra at 25°C and the fluorescence spectra at 77°K. The –¹⁴CO₂ fixation was inhibited by 80–90% in treated leaves. The fluorescence induced by the measuring light was greater in the treated leaves than in the control ones, and the kinetics of the decline of the relative fluorescence intensity were also different. Electron microscopic studies showed macrogranum-like structures and incomplete membrane vesicles in the treated plastids. After longer treatment a destruction of membranes was observed. The results indicate some structural and functional membrane deficiencies and instability of the membranes.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

Cloning and characterization of the phosphatidylserine synthase gene of Agrobacterium sp. strain ATCC 31749 and effect of its inactivation on production of high-molecular-mass (1-->3)-beta-D-glucan (curdlan)

Genes involved in the production of the extracellular (1-->3)-beta-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pss(AG), encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the Pss(AG) protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pss(AG)::TnphoA mutation was complemented by the intact pss(AG) gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.