- Volume 140, Issue 2, 1994

Summary: The interrelationship of the multiple cysteine proteases (CPs) found characteristically at high activity in Leishmania mexicana amastigotes has been investigated. The mature forms of the five enzymes of groups B and C, which have subtly different substrate preferences, are the same size. Enzymically deglycosylated group A CP proteins also have the same molecular mass. Proteases of all three groups are specifically recognized by antisera raised against the group B or group C CPs. In addition, CPs of groups A, B and C have highly similar N-terminal amino acid sequences. The consensus sequence matches that predicted from the sequenced Imcpb gene, which occurs in a tandem array of over ten similar genes. Thus, the results are consistent with the groups A, B and C CPs being products of different Imcpb genes within the array, the different genes encoding CPs with identical N-termini, but with limited amino acid substitutions within the mature enzyme accounting for the different properties of the CPs. Evidence is also presented to indicate membrane-association of proteolytically active but less processed forms of Imcpb products.

Summary: The toxin produced by Pichia acaciae was purified and its properties comp to those of the toxin from Kluyveromyces lactis. Like this toxin, the P. aca toxin is a protein comprised of three subunits (molecular masses 110, 39 a 38 kDa) with an associated chitinase activity and a pH optimum between 7.0 and 7.5. P. acaciae toxin also caused G1 cell cycle arrest. Of the thirteen recessive alleles that provided resistance in Saccharomyces cerevisiae to A lactis toxin, only three also conferred resistance to P. acaciae toxin. Simila and differences in the interactions of the two toxins with yeast cells are discussed.

Summary: Cells of the dimorphic yeast Candida albicans are easily induced to germinate in synchrony. Using germinating cells of strain FC18, we examined the effects of several drugs that are known to affect the cytoskeleton on growth and cytoskeletal organization. Cytochalasin A (CA), an inhibitor of actin function, inhibited the germination of the yeast cells and changed the cylindrical expansion of the apex of the germ tube to swelling growth. Effects of CA on the organization of actin were examined with rhodamine-phalloidin (Rh-Ph), which specifically stains F-actin. In CA-untreated cells, Rh-Ph staining resulted in condensed dot-like fluorescence at the growing tip, as well as filamentous fluorescence (actin cables) that ran from the apex to the basal region. In CA-treated cells, condensed dot-like fluorescence was still observed at the swelling tip, but actin cables had disappeared completely. This result indicates that CA does not affect the asymmetrical distribution of actin, and suggests that the actin cables are not required for maintenance of the polarized localization of actin. Benomyl, an anti-microtubule drug, inhibited the germination of yeast cells and the apical growth of germinated cells. Benomyl not only disrupted microtubules (MTs), but also affected the distribution of actin. In benomyl-treated cells, actin dots were randomly dispersed all over the cell. This result indicates that benomyl destroyed the mechanism that maintains the asymmetrical distribution of actin, and suggests that MTs are involved in such a mechanism. The polarized localization of organelles is one of the most important factors associated with dimorphism. Our data suggest that the cytoskeleton, composed of actin and MTs, is involved in the control of polarity in the hyphal growth of C. albicans, and that actin and MTs are interrelated in the establishment of polarity.

Summary: Yeast cells of Candida albicans produced germ tubes in a salt-glucose medium containing 4% calf serum at pH 7 and 37 °C. Hyphal growth continued for 24 h and the filaments did not revert to yeast cells. When cells were grown at pH 4, reversion to yeast growth was observed, despite the presence of serum. The elongation of hyphae was inhibited within 30 min. The distribution of microtubules and microfilaments during pH-regulated morphological transition was studied by an immunofluorescence technique using an antitubulin antibody with a FITC-conjugated secondary antibody, and by staining with tetramethylrhodaminyl phalloidin for filamentous actin and actin granules. After changing to acidic conditions, microtubules were distributed normally in the cytoplasm; however, microfilaments disappeared from hyphal cells, and actin granules were localized at the site of budding. These results show that microfilaments play an important role during pH-regulated morphological transition.

Summary: A simple and efficient protoplast regeneration and PEG-induced transformation procedure was developed for Bacillus licheniformis. The protoplast stabilizing osmoticum was 0.5 M sucrose in the regenerating media, and the restoration of cell wall was carried out at pH 8.4. The mean frequency of regeneration was 83%. The transformation efficiency was studied with four different strains and five plasmids, and ranged from 3.5×105to 7.2×106transformants per μg plasmid DNA depending on the plasmid, and on the donor and recipient strains used. Endonuclease digested and ligated plasmid DNA could also be used to transform the protoplasts, but at a lower frequency.

Summary: From a partial Sau3AI library of Streptomyces coelicolor A3(2) DNA in pIJ916, two hybrid plasmids pGX1 and pGX2 were isolated that complemented S. coelicolor A3(2) or S. lividans arginine auxotrophs. Subcloning DNA from pGX1 in the Escherichia coli expression vector pRK9 containing the Serratia marcescens trp promoter gave rise to one plasmid, pZC2, that complemented E. coli argB, C, E and H auxotrophs, and another, pZC1, that complemented only the first three. The plasmids were markedly unstable in the various complemented hosts, to varying extents; pZC1 was characterized further as providing the stablest host/plasmid combinations. In vitro deletion of part of the vector's trp promoter did not affect complementation of the argB and C auxotrophs, implying that the S. coelicolor A3(2) arg genes may be expressed from their own promoter. The trp promoter-less plasmids included isolates, such as pZC177, that had suffered extensive further deletion without loss of complementing ability. Extracts of an E. coli argE auxotroph carrying pZC177 showed ornithine acetyltransferase activity, indicating that the complementing gene is of the argJ type. The complementation properties of in vitro deletion derivatives of pZC177 indicated the gene order argC-J-B. Part of argC and the upstream region were sequenced; an ORF was identified whose predicted product showed appreciable homology with the E. coli and Bacillus subtilis ArgC polypeptide. Upstream of this ORF a consensus-type promoter and ribosome binding site could be discerned; overlapping its promoter was a sequence with homology to arginine operators in these two other organisms. An in vitro frameshift in argC had a polar effect on expression in E. coli of argJ and B, suggesting that the three genes are transcribed in the same direction, possibly as an operon.

Summary: In cell-free extracts of Escherichia coli clones harbouring the 3.5 kb BglII fragment of plasmid pTC1 from Rhodococcus rhodochrous CTM a catechol 2,3-dioxygenase (C230) accepting both 3-methylcatechol and 2,3-dihydroxy-biphenyl as substrates could be detected. The plasmid-encoded gene for C230 of R. rhodochrous CTM and its flanking regions were sequenced. In front of the gene a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The derived amino acid sequence of the C230 was compared to that of nine other enzymes, which all catalyse the extradiol cleavage of an aromatic ring. These nine sequences were from different Pseudomonas strains, in contrast to the sequence described here, from a Gram-positive bacterium. The role of four strongly conserved histidines was examined by chemical modification of the histidyl residues of the native enzyme by diethylpyrocarbonate. For that purpose the C230 was purified to homogeneity from E. coli harbouring pSC1701. However, the enzyme lost its activity during the purification. Activity could partially be restored by treatment with Fe2+and reducing agents.

Summary: Erwinia herbicola is known to synthesize carotenoids and gives an orange-coloured phenotype. These carotenoids play a role in the protection of the cells from the damage caused by near-UV irradiation in nature. The genes encoding these carotenoids in E. herbicola Eho13 are clustered in a 7 kb DNA fragment. The complete sequence of this fragment has been determined. DNA sequence analysis revealed that the entire sequence contains at least five genes, which are transcribed in the same direction. These five genes are organized in the order crtE-crtX-crtY-crtI-crtB. A gene fusion study showed that two different regions in this 7 kb gene cluster contain promoter activity. Primer-extension analysis identified two transcription start sites, located 147 bp upstream from the first gene of the cluster, crtE, and within the last gene of the cluster, crtB. An RNA-PCR study suggested that the five crt genes were organized in an operon and were transcribed from the promoter upstream from crtE.

Summary: Salmonella typhimurium encounters a variety of acid conditions during both its natural and pathogenic existence. The ability of this organism to respond transcriptionally to low pH is an area of active interest but little knowledge. As part of an ongoing investigation of low-pH adaptation, 18 pH-controlled IacZ operon fusions in Salmonella typhimurium have been identified (15 in this study) and categorized into at least 11 different loci. They include iroA (at 57 min), aciA (99 min), aciB (90-93 min), aciD (ompC, 45 min), aciJ, aciK(33-36 min), aniC (93 min), anil (33-36 min), hyd (59 min), cadA (54 min) and aniG (63 min). All but two were induced by low pH. One of the exceptions, the iron-regulated iroA locus, was induced at high pH. The unusual aciA locus was induced by low pH under semiaerobic conditions but high pH under aerobic conditions. Most of the other aci genes were expressed best under anaerobic conditions. Many of these genes exhibited strict co-inducer requirements for small molecules to be expressed in minimal medium. These included iron for iroA, tyrosine for aniC, I and aciK, mannose for aniG, formate for hyd, lysine for cadA, and unknown components of complex medium for aciA, aciB and aciD. Six regulatory circuits were revealed involving at least five regulatory loci (fur, oxrG, earAB, earC and ompR). As part of the adaptive response to low pH, S. typhimurium will induce an acid protection system called the acid tolerance response (ATR). As has been shown for fur mutations, the oxrG regulatory mutation interfered with the normal induction of this system.

Summary: A 2.5 kb DNA fragment of the Saccharomyces cerevisiae SYR1 gene was cloned by complementation of the syr1 mutations that simultaneously lead to resistance to the phytotoxin syringomycin and sensitivity of growth to high Ca2+concentrations. Sequencing of this fragment revealed a single open reading frame encoding a polypeptide of 365 amino acids. Four hydrophobic regions each separated by hydrophilic regions were present in the protein. SYR1 was identical to ERG3, which is suggested to encode C-5 sterol desaturase required for ergosterol biosynthesis. The protein product of SYR1 was identified by Western blot analysis as a protein of 40 kDa in the particulate fraction. Gene disruption experiments demonstrated that elimination of SYR1/ERG3 is not lethal, but results in membrane C-5 desaturated sterol deficiencies, resistance to syringomycin and sensitivity to high Ca2+. The syr1 mutant cells had significantly decreased ability for syringomycin binding. The results indicated that C-5 desaturated sterols are involved in the binding of syringomycin to the cell, and the lack of the sterols in the mutant membrane results in sensitivity to high Ca2+and an increased rate of cellular Ca2+influx.

Summary: Sakacin P is a heat-stable, unmodified peptide bacteriocin produced by Lactobacillus sake LTH 673. The strain was isolated from fermented dry sausages and is well adapted to this habitat. The bacteriocin inhibits the growth of the opportunistic food pathogens Enterococcus faecalis and Listeria monocytogenes and therefore, it may improve the hygienic status of fermented food, i.e. meat products. Oligonucleotide probes were designed from the N-terminal amino acid sequence of sakacin P and used to identify sakP, the structural gene of sakacin P, on the chromosome of L. sake LTH 673. SakP was cloned into Escherichia coli NM554 and the nucleotide sequence of the gene and its adjacent regions were determined. Sakacin P appears to be synthesized as a prepeptide of 61 amino acids which is proteolytically processed to the mature bacteriocin consisting of 43 amino acids. Sequencing of the cloned fragment also revealed the presence of two other open reading frames orfX and orfY, which are located upstream and downstream of sakP, respectively, putatively encoding proteins of 52 and 98 amino acids, respectively. The functions of both ORFs remain unknown. Primer extension analysis revealed a promoter upstream of sakP. Two transcripts of approximately 0.35 and 1.0 kb were detected by Northern hybridization encoding either only sakP, or both sakP and orfY, respectively.