- Volume 144, Issue 12, 1998

Summary: The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants. This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures. The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells. Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature. The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant. Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins. This new allele was named cdc10-11. The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate. Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 °C, although the morphogenetic phenotype at 37 °C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area.

Summary: Fission yeast cells grow by extension at the ends (poles) and divide by transverse fission. It has previously been reported that Schizosaccharomyces japonicus var. japonicus can switch to unipolar, filamentous growth. Here it is shown that the yeast-to-mycelium transition is a gradual process involving a changeover to unipolar growth associated with asymmetric divisions, the development of large polarly located vacuoles, the modifications of the actin and microtubular cytoskeleton and the repression of cell separation after division. High concentrations of glucose in the medium or supplementation of the medium with caffeine or cAMP support the bipolar yeast phase, inhibit the transition to the mycelial phase and induce the conversion of hyphae to yeasts. These effects suggest that cAMP may be involved in the regulation of dimorphism. Temperatures below 18 °C or over 35 °C are restrictive for the mycelial phase and provoke a return to yeast phase.

Summary: Streptomycin dependence can be caused by mutations in ribosomal protein S12. Mutations suppressing such streptomycin dependence have been found in ribosomal proteins S4 and S5, and in 16S rRNA. Here a new suppressor mutation localized in elongation factor Tu (EF-Tu) is described, consistent with recent models of ribosome-EF-Tu-tRNA interaction at the decoding centre. The EF-Tu mutation was obtained by genetic selection for streptomycin independence; it was identified as Ala375 → Thr, previously described as EF-TuAR and known to confer a kirromycin-resistant, error-prone phenotype. Also, other streptomycin-dependent (SmD) S12 mutations could be complemented by this mutation. The streptomycin-independent (SmI) strain grows more slowly than the wild-type (wt), suggesting that not all the defects of the S12 mutation can be complemented by EF-Tu[A375T]. Moreover, this strain is more susceptible than wt to reduction in the cellular EF-Tu concentration, and disruption of tufB led to considerable growth-rate impairment. Expression of EF-Tu from tufB, not only of wt EF-Tu and EF-Tu[A375T] but, remarkably, also of EF-Tu[G222D], known as EF-TuB0 and defective in protein synthesis, equally contributed to cell growth. In vitro analysis revealed a decreased translational activity of wt EF-Tu with SmD ribosomes as compared to EF-Tu[A375T], while EF-Tu[G222D] showed no activity at all, just as with wt ribosomes. Possible mechanisms are discussed for the improved growth rate observed in such SmI strains when they include wt EF-Tu or EF-Tu[G222D].

Summary: The Escherichia coli maltose regulon consists of five operons under the control of the MalT transcriptional activator. lac operon fusions were constructed in vitro with the MalT-dependent promoter and with the malT promoter itself. -Galactosidase activity displayed by these fusions during growth at different external pH (pHo) revealed that growth at a pHo higher than 6 stimulates the transcription of malT- and MalT-controlled genes in the absence or presence of maltose. Using a malTp1 malTp10 promoter that is cAMP-CRP (cAMP receptor protein)-independent, it was demonstrated that CRP is essential for malT pHo regulation and that the pHo-dependent activity of malKp is a direct consequence of malT regulation. The pHo regulation displayed by a deleted but still functional malT promoter fused to lacZ demonstrates that this minimal promoter contains all the regulatory regions for establishing pHo regulation. In the absence of Mlc, a repressor of malT expression, the pHo regulation of malT was still effective. It is proposed that binding of cAMP-CRP at malTp may be affected by malTp topology induced by pHo or that a pHo-dependent effector may act in concert with the cAMP-CRP complex.

Summary: The temperate bacteriophage VWB integrates into the chromosome of Streptomyces venezuelae ETH14630 via site-specific integration. Following recombination of the VWB attP region with the chromosomal attB sequence, the host-phage junctions attL and attR are formed. Nucleotide sequence analysis of attP, attB, attL and attR revealed a 45 bp common core sequence. In attB this 45 bp sequence consists of the 3' end of a putative tRNAArg(AGG) gene with a 3'-terminal CCA sequence which is typical for prokaryotic tRNAs. Phage DNA integration restores the putative tRNAArg(AGG) gene in attL. However, following recombination the CCA sequence is missing as is the case for most Streptomyces tRNA genes described so far. Adjacent to VWB attP, an ORF encoding a 427 aa protein was detected. The C-terminal region of this protein shows high similarity to the conserved C-terminal domain of site-specific recombinases belonging to the integrase family. To prove the functionality of this putative integrase gene (int), an integrative vector pKT02 was constructed. This vector consists of a 23 kb HindIII-Sphl restriction fragment of VWB DNA containing attP and int cloned in a non-replicative Escherichia coli vector carrying a thiostrepton-resistance (tsr) gene. Integration of pKT02 was obtained after transformation of Streptomyces venezuelae ETH14630 and Streptomyces lividans TK24 protoplasts. This vector will thus be useful for a number of additional Streptomyces species in which a suitable tRNA gene can be functional as integration site.

Summary: Trichodesmium spp. are marine filamentous, non-heterocystous cyanobacteria capable of aerobic nitrogen fixation. In this study, the nitrogenase structural genes (nifHDK) and nifU gene of Trichodesmium sp. IMS101 were cloned and sequenced. The Trichodesmium sp. IMS101 nifH, nifD and nifK amino acid sequences showed only 79%, 66% and 68% identity, respectively, to those of Anabaena sp. strain PCC 7120. A potential transcription start site for nifH was found 212 bases upstream of the nifH start codon. Promoter-like nucleotide sequences upstream of the transcription start site were identified that were very similar to those identified for the nitrogenase genes of Anabaena spp. Sequence analysis revealed regions of DNA that may form stem-loop structures in the intercistronic regions downstream of nifH and nifD. RNA analysis by Northern hybridization revealed the presence of transcripts corresponding to nifH, nifHD and nifHDK. Surprisingly, Northern hybridization also revealed the presence of transcripts that corresponded to nifD, nifDK and nifK, which have not been previously reported as transcripts in contiguous nifHDK genes of cyanobacteria. Transcription of the nifHDK genes was not significantly repressed in the presence of nitrate at a final concentration of 20 mM or at oxygen concentrations of up to 40%, whereas ammonium and urea inhibited nifHDK transcription. The transcription of the nifHDK genes was not affected by darkness, which suggests that transcription of these genes in Trichodesmium is not directly regulated by light.

Summary: The sector of the genome of broad-host-range IncP plasmid RK2 from kb coordinate 54·0 to 60·0 confers an active partitioning phenotype, increasing the segregational stability of low-copy-number unstable plasmids. This Par region encodes the central control operon (korA, incC, korB, korF and korG) and the associated genes kfrA, upf54.8 and upf54.4. Each ORF in this region was knocked out in turn and it was shown that only incC and korB are needed for the stability phenotype. incC encodes two polypeptides from alternative translational starts. A deletion of the start of the operon showed that only IncC2, the shorter product is essential for partitioning. Directed mutation or deletion was used to inactivate in turn each of the three KorB-binding sites (Obs) which were candidate cis-acting sequences needed for stability. Only inactivation of OB3, which lies between upf54.4 and upf54.8, resulted in an increased rate of segregational loss. However, the rate of loss was significantly higher than the rate of loss of the test plasmid carrying none of this RK2 Par region. Either inactivation of korB or deletion of Ob1 from this Ob3 mutant resulted in restoration of the loss rate to that expected for the unstable test plasmid alone. Thus KorB can act on Ob1 to create a complex that either inhibits replication or reduces the effective plasmid copy number, perhaps by promoting pairing between plasmid molecules. This implies that RK2 goes through a cycle of pairing and separation, akin to the mitotic cycle of eukaryotic chromosomes.

Summary: Protein secretion in Gram-negative bacteria is often dependent on the general secretory pathway (GSP). In Pseudomonas aeruginosa, this system requires at least 12 Xcp (Gsp) proteins, which are proposed to constitute a multiprotein complex localized in the bacterial envelope. Hitherto, little was known about the mutual interactions between Xcp proteins. In this study, mutants affected in the xcpZ gene encoding a bitopic inner-membrane protein were analysed to investigate the role of this protein in the architecture of the secretory machinery. The absence of XcpZ resulted in a decreased amount of XcpY. Reciprocally, XcpZ was not detectable in a xcpY mutant demonstrating a mutual stabilization of these two proteins. These results strongly suggest that XcpZ and XcpY interact within the functional secretory apparatus.

Summary: Three new genes encoding the serine-aspartate (SD) repeat-containing proteins SdrC, SdrD and SdrE were found in Staphylococcus aureus strain Newman. The SD repeats had earlier been found in the S. aureus fibrinogen-binding clumping factors ClfA and ClfB. The clfA and clfB genes encode high-molecular-mass fibrinogen-binding proteins that are anchored to the cell surface of S. aureus. The sdr genes now reported are closely linked and tandemly arrayed. The putative Sdr proteins have both organizational and sequence similarity to ClfA and ClfB. At the N-terminus, putative secretory signal sequences precede approximately 500 residue A regions. The A regions of the Sdr and Clf proteins exhibit only 20–30% residue identity when aligned with any other member of the family. The only conserved sequence is the consensus motif TYTFTDYVD. The Sdr proteins differ from ClfA and ClfB by having two to five additional 110–113 residue repeated sequences (b-motifs) located between region A and the r-region. Each b-motif contains a consensus Ca2+-binding ef-hand loop normally found in eukaryotic proteins. The structural integrity of recombinant Sdrd(B1-B5) protein comprising the five b-repeats of SdrD was shown by bisANS fluorescence analysis to be Ca2+-dependent, suggesting that the ef-hands are functional. When Ca2+was removed the structure collapsed to an unfolded conformation. The original structure was restored by addition of Ca2+. The C-terminal r-domains of the Sdr proteins contain 132–170 sd residues. These are followed by conserved wall-anchoring regions characteristic of many surface proteins of Gram-positive bacteria. The Sdr locus was present in all 31 S. aureus strains from human and bovine sources tested by Southern hybridization, although in a few strains it contained two rather than three genes.

Summary: Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5′ end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3′ end of the tRNAAlagene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNAAlagene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.

Summary: The structure and distribution of IS990, a new Mycobacterium tuberculosis DNA sequence with homology to characterized insertion sequences (ISs), were investigated. IS990 was related to IS elements of the IS3 family and was present as a single copy in all 21 investigated M. tuberculosis strains, two Mycobacterium bovis strains and two M. bovis BCG strains. The sequence appears to be specific for the M. tuberculosis complex. The element carries two frameshift mutations and appears to be defective.

Summary: The technique of representation difference analysis PCR has been applied to find genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp fragment which was used to isolate a larger genetic element found in the enteric pathogens M. avium subsp. paratuberculosis and M. avium subsp. silvaticum but which was absent from the very closely related and relatively benign M. avium subsp. avium. This element, designated GS, is greater than 6·5 kbp in length and has a G+C content 9 mol% lower than other genes from this species. There is a previously uncharacterized insertion sequence associated with one end. The GS element encodes five ORFs in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum, all of which have counterparts encoded in Mycobacterium tuberculosis. Database searches revealed homologues for these ORFs in a number of bacterial species, predominantly Gram-negative organisms, including a number of enteric pathogens. These homologous genes encode functions related to LPS or extracellular polysaccharide biosynthesis. This element has a number of features in common with pathogenicity islands such as its low G+C content, an association with a putative insertion sequence and a grouping of genes of related function with a possible link to virulence. No direct link to pathogenicity has been shown but GS may belong to a group of related ‘genetic islands’ and represents the first such element to be identified in mycobacteria.