- Volume 148, Issue 9, 2002

Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269·6, 1298·5, 1312·5 and 1326·5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2·5 μg ml−1 against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC50 of 4·5±0·07 ng ml−1. This report also describes the potential of the munumbicins in medicine and agriculture.

The cloning of a gene encoding the novel phosphotriesterase from Pseudomonas monteilii C11, which enabled it to use the organophosphate (OP) coroxon as its sole phosphorus source, is described. The gene, called hocA (hydrolysis of coroxon) consists of 501 bp and encodes a protein of 19 kDa. This protein had no sequence similarity to any proteins in the SWISS-PROT/GenBank databases. When a spectinomycin-resistance cassette was placed in this gene, phosphotriesterase activity was abolished and P. monteilii C11 could no longer grow with coroxon as the sole phosphorus source. Overexpression and purification of HocA as a maltose-binding protein fusion produced a protein having a broad substrate specificity across oxon and thion OPs. Michaelis–Menten kinetics were observed with the oxon OPs, but not with the thion OPs. End-product inhibition was observed for coroxon-hydrolytic activity. Increased expression of hocA was observed from an integrative hocA–lacZ fusion when cultures were grown in the absence of phosphate, suggesting that it might be part of the Pho regulon, but the phosphate-regulated promoter was not cloned in this study. This is believed to be the first study in which a gene required for an organism to grow with OP pesticides as a phosphorus source has been isolated.

The methylotrophic yeast Candida boidinii exhibits formaldehyde dehydrogenase activity (FLD, EC 1.2.1.1) during growth on methanol as a sole carbon source. The structural gene, FLD1, was cloned from a genomic library of C. boidinii. The 1263 bp FLD1 gene contained a 123 bp intron and its exon encoded a gene product of 380 amino acids, whose predicted amino acid sequence showed high similarity to the sequences of FLDs from other organisms. The FLD1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The fld1Δ strain could not grow on methanol as a carbon source under methanol-limited chemostat culture conditions, even with low dilution rates (D<0·05 h−1), whereas a strain with a disruption in the gene for formate dehydrogenase (FDH; another NADH-generating dehydrogenase involved in the formaldehyde oxidation pathway) could survive. These results indicated that FLD, but not FDH, is essential for growth of C. boidinii on methanol.

Intracellular pathogens have developed different mechanisms which enable their survival and replication within the host cells. Some survive and replicate within a membrane-bound vacuole modified by the bacteria to support microbial growth (e.g. Salmonella enterica serovar Typhimurium), whereas others escape from the vacuole into the host cell cytosol, where they proliferate (e.g. Listeria monocytogenes). In this study a Salmonella strain carrying a mutation in sifA which is released from the vacuole was used to analyse Salmonella survival and replication within the cytosol of several cell lines. It was found that Salmonella replicates within the cytosol of epithelial cells at a higher rate than that achieved when replicating within the vacuole, but is defective for replication in the cytosol of fibroblasts or macrophages. Using an aroC purD double mutant strain which does not replicate within host cells, it was shown that Salmonella encounters a killing activity within the cytosol of macrophages. Furthermore, in vitro experiments using cytosol extracted from either infected or uninfected macrophages suggested that this activity is activated upon Salmonella infection.

The possibility that nonculturable cells of a normally culturable bacterial pathogen may constitute a source or reservoir for infective disease was investigated. In multiple experiments and with careful attention to the statistical limitations of the assays used, Salmonella enterica serovar Typhimurium cells rendered nonculturable by carbon and nitrogen stress in the presence of chloramphenicol were administered orally and intraperitoneally to over 300 female BALB/c mice. Neither infection nor colonization was detected in these studies, even when active but nonculturable (ABNC) cells, as defined by the Kogure cell elongation assay, were present in the inoculum. Doses of ABNC cells exceeding the oral and intraperitoneal LD50 values by 3·5 and 2 orders of magnitude, respectively, were administered. It was concluded that ABNC cells of the salmonella strains used could not be considered potentially infective and that their detection in samples from material being evaluated as a potential source or reservoir of infection by the Kogure test does not specifically represent an infective hazard.

Components of the ATP-dependent Clp protease complex are found in a wide range of prokaryotic cells and they are often expressed as part of the cellular stress response. To investigate the physiological role of the proteolytic subunit, ClpP, in Salmonella enterica serovar Typhimurium (S. typhimurium) an in-frame deletion of the clpP gene was constructed. Growth experiments revealed that clpP is important for the ability of S. typhimurium to grow under various stressful conditions, such as low pH, elevated temperature and high salt concentrations. Since the stationary-phase sigma factor, RpoS, is a target of the Clp proteolytic complex, the effect of the clpP deletion in the absence of RpoS was examined; it was observed that growth of the S. typhimurium clpP mutant is affected in both an RpoS-dependent and an RpoS-independent manner. Analysis of the degradation of abnormal puromycyl-containing polypeptides showed that ClpP participates in the proteolysis of such proteins in S. typhimurium. These findings prompted an investigation of the growth of an Escherichia coli clpP mutant under various stress conditions. The growth of this E. coli mutant was affected by heat, salt and low pH, although not to the same extent as observed for the S. typhimurium clpP mutant. The results of this study indicate that the S. typhimurium clpP mutant is generally more sensitive to environmental stress than the E. coli clpP mutant and it is proposed that this is due to a reduced ability to degrade misfolded proteins generated under these conditions.

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhoea in young children. The locus of enterocyte effacement (LEE) pathogenicity island comprises a cluster of operons encoding a type III secretion system and related proteins that are associated with EPEC virulence. The LEE1 operon encodes Ler that positively regulates the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units. The LEE operons are repressed at 27 °C and expressed at 37 °C. This paper describes a regulatory cascade of the thermoregulation of LEE operons. LEE1 including ler is repressed by H-NS at 27 °C but not at 37 °C. In contrast, the expression of the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units is repressed by H-NS at both 27 °C and 37 °C. Upon shifting the culture temperature from 27 °C to 37 °C, Ler is synthesized and in turn activates the expression of LEE2, LEE3, LEE4 and espG by releasing the H-NS mediated repression. In the case of LEE5, Ler acts both by alleviating the H-NS mediated repression and by an additional mechanism, as yet to be defined.

Characteristics of Escherichia coli residing in the vagina and their role in extraintestinal infections are largely unknown. In this study, 88 vaginal E. coli (VEC) isolates from Japanese women were characterized by extraintestinal virulence factor (VF) profiling, O:H serotyping and phylogenetic analysis. The prevalence of papC, hlyA, cnfI, PAI, ibeA and K1 antigen among the VEC strains were 45, 22, 19, 78, 32 and 44%, respectively. Phylogenetic analysis identified 76, 16 and 8% of the VEC strains in groups B2, D and A, respectively. The VEC strains were distributed into 31 serotypes, including 8 common serotypes (O1:K1:H1, O1:K1:H7, O2:K1:H7, O4:H5, O6:H1, O18ac:K1:H7, O25:H1 and O75:HNM) that were identified in three or more isolates. Comparative analysis with 61 stool isolates from healthy Japanese men and women, and with data from previous studies, revealed that, although some geographical specificities do exist, the VEC strains shared common VF profiles, O:K:H serotypes and phylogeny with uropathogenic E. coli and E. coli of neonatal septicaemia and meningitis. This study provides additional evidence for a link among extraintestinal E. coli, supporting the concept that the VEC are a reservoir along the ‘faecal–vaginal–urinary/neonatal’ course of transmission in the extraintestinal E. coli infections.

Campylobacter jejuni is a food-borne pathogen responsible for infectious enterocolitis. The early-response transcription factor NF-κB triggers the expression of genes associated with cellular immune and inflammatory responses. Co-incubation of HeLa cells with viable C. jejuni leads to the activation of the transcription factor NF-κB as determined by specific induction of a cellular luciferase-based reporter. Boiled cell-free extracts of C. jejuni are also potent dose-dependent stimulators of NF-κB-dependent transcription, the levels of which can reach up to 1000-fold as compared with independent controls. Using both cultured HeLa cells and human colonic epithelial (HCA-7) cells, the activation of NF-κB by C. jejuni boiled extract has been monitored through the degradation of IKBα and DNA binding of the nuclear translocated p50/p65 heterodimer of NF-κB. These events are co-ordinated with elaboration of the pro-inflammatory cytokine interleukin-8. Fractionation of the boiled C. jejuni extract suggests that the majority of the bioactive component has a molecular mass of 3 kDa or less, which is insensitive to proteinase K treatment.

Proteomics is a powerful tool for analysing differences in gene expression between bacterial strains with alternate phenotypes. Staphylococcus aureus strains are grouped on the basis of their sensitivity to methicillin. Two-dimensional gel electrophoresis was combined with MS to compare the protein profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 ‘hypothetical’ proteins was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain. Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence of TX-100, 44 protein spots showed altered levels of abundance, and 11 of these spots were found only in COL. The products of genes regulated by σB (the alternative sigma factor), including Asp23 and three proteins of unknown function, and SarA (a regulator of virulence genes) were shown to be present at significantly altered levels. SarA production was induced in TX-100-treated cultures. A protein of the σB operon, RsbV, was only detected in COL and its production was down-regulated in COL when the strain was treated with TX-100, whereas RsbW was present at reduced levels in both strains. Upon growth of both strains in the presence of TX-100, no effects on the production of the essential methicillin-resistance factor FemA were detected, whereas phosphoglucosamine mutase (GlmM) production was reduced in COL alone. This study suggests that proteins of the σB and sarA regulons, as well as other factors, are involved in methicillin resistance in S. aureus.

For an economically feasible production of ethanol from plant biomass by microbial cells, the fermentation of xylose is important. As xylose uptake might be a limiting step for xylose fermentation by recombinant xylose-utilizing Saccharomyces cerevisiae cells a study of xylose uptake was performed. After deletion of all of the 18 hexose-transporter genes, the ability of the cells to take up and to grow on xylose was lost. Reintroduction of individual hexose-transporter genes in this strain revealed that at intermediate xylose concentrations the yeast high- and intermediate-affinity transporters Hxt4, Hxt5, Hxt7 and Gal2 are important xylose-transporting proteins. Several heterologous monosaccharide transporters from bacteria and plant cells did not confer sufficient uptake activity to restore growth on xylose. Overexpression of the xylose-transporting proteins in a xylose-utilizing PUA yeast strain did not result in faster growth on xylose under aerobic conditions nor did it enhance the xylose fermentation rate under anaerobic conditions. The results of this study suggest that xylose uptake does not determine the xylose flux under the conditions and in the yeast strains investigated.

The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS). Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups. Mutation in the β-subunit of F1-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA (ρ0 mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-xL, when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity. However, this co-expression did not prevent the production of ROS. These data suggest that in K. lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-xL is not limited to its antioxidant property.

The heterotrimeric G-protein α-subunit family plays multiple roles in eukaryotic cells, such as the regulation of growth and development, of pathogenicity and of the transmission of pheromone stimulation. In the homobasidiomycete Schizophyllum commune, some genes encoding heterotrimeric G-protein α-subunits (SCGPα1, SCGPα2, ScGP-A, ScGP-B and ScGP-C) have been reported. In this study, constitutively active mutants of ScGP-A, ScGP-B and ScGP-C were generated by site-directed mutagenesis and introduced into the S. commune monokaryon strain to investigate the function of each gene. Northern analysis showed that the mutated genes were strongly expressed when compared with endogenous G-proteins in many clones. Upon macroscopic examination, some transformed clones expressing ScGP-A (Q207R) and ScGP-C (Q204R) mutant genes exhibited a slight suppression of aerial-hyphae formation in the monokaryon strain. In contrast to the slight suppression of aerial-hyphae formation in the monokaryon, most clones expressing mutated ScGP-A or ScGP-C genes failed to form fruit-bodies in the dikaryon strain. This observation indicated that ScGP-A and ScGP-C played a role in suppressing fruit-body formation in the dikaryon. Furthermore, these phenotypes were similar to the phenotype of the thn mutant in S. commune to some extent. Since the thn-1 gene encodes a putative regulator of the G-protein signalling protein (RGS), ScGP-A and ScGP-C might be targets of thn-1.

Numerous chitin synthase structural (CHS) genes have been identified in fungi, and usually there are several CHS genes per species. Compensatory expression of one CHS gene in response to defects in other CHS genes has not been reported. Five chitin synthase structural (WdCHS) genes have been identified in the melanized human pathogen Wangiella dermatitidis: WdCHS1, WdCHS2, WdCHS3, WdCHS4 and WdCHS5. This study showed that increased WdCHS expression existed as a compensatory mechanism in response to stress induced by chitin synthase gene disruptions, or by exposure of the wild-type or two temperature-sensitive morphological mutants, for short or long periods, to 37 °C. In general, the compensatory responses varied with each WdCHS gene, and in accordance with the hypothesized functions of the chitin synthase (WdChsp) encoded. It is suggested that these compensatory responses indicate that WdCHS gene transcription in W. dermatitidis functions as part of a cell-wall integrity pathway in a manner similar to that recently described for Saccharomyces cerevisiae.

In the filamentous fungus Aspergillus fumigatus, the vast majority of the cell-wall-associated proteins are secreted proteins that are in transit in the cell wall. These proteins can be solubilized by detergents and reducing agents. Incubation of a SDS/β-mercaptoethanol-treated cell-wall extract with various recombinant enzymes that hydrolyse cell-wall polysaccharides resulted in the release of a unique protein in minute amounts only after incubation of the cell wall in the presence of 1,3-β-glucanase. Sequence analysis and biochemical studies showed that this glycoprotein, with an apparent molecular mass of 80 kDa, was an acid phosphatase (PhoAp) that was active on both phosphate monoesters and phosphate diesters. PhoAp is a glycosylphosphatidylinositol-anchored protein that was recovered in the culture filtrate and cell-wall fraction of A. fumigatus after cleavage of its anchor. It is also a phosphate-repressible acid phosphatase. The absence of PhoAp from a phosphate-rich medium was not associated with a reduction in fungal growth, indicating that this cell-wall-associated protein does not play a role in the morphogenesis of A. fumigatus.

Type II methane-oxidizing bacteria (MOB) were isolated from diverse environments, including rice paddies, pristine and polluted freshwaters and sediments, mangrove roots, upland soils, brackish water ecosystems, moors, oil wells, water purification systems and livestock manure. Isolates were identified based on morphological traits as either Methylocystis spp., Methylosinus sporium or Methylosinus trichosporium. Molecular phylogenies were constructed based on nearly complete 16S rRNA gene sequences, and on partial sequences of genes encoding PmoA (a subunit of particulate methane monooxygenase), MxaF (a subunit of methanol dehydrogenase) and MmoX (a subunit of soluble methane monooxygenase). The maximum pairwise 16S rDNA difference between isolates was 4·2%, and considerable variability was evident within the Methylocystis (maximum difference 3·6%). Due to this variability, some of the published ‘specific’ oligonucleotide primers for type II MOB exhibit multiple mismatches with gene sequences from some isolates. The phylogenetic tree constructed from pmoA gene sequences closely mirrored that constructed from 16S rDNA sequences, and both supported the presently accepted taxonomy of type II MOB. Contrary to previously published phylogenetic trees, morphologically distinguishable species were generally monophyletic based on pmoA or 16S rRNA gene sequences. This was not true for phylogenies constructed from mmoX and mxaF gene sequences. The phylogeny of mxaF gene sequences suggested that horizontal transfer of this gene may have occurred across type II MOB species. Soluble methane monooxygenase could not be detected in many Methylocystis strains either by an enzyme activity test (oxidation of naphthalene) or by PCR-based amplification of an mmoX gene.

The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined. The G+C content of the sequence of this plasmid was 62 mol%. Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44·1 and 37·8 kDa, respectively. Mutational analysis showed that ORF2 was crucial for plasmid replication. The translational product of ORF2 shared local homologies with replication proteins of several θ-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts. An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV). ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria. A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli. MOB modules of pWKS1 and pBBR1 were functionally complementary to each other. Hybridization analysis revealed that only plasmid pSOV1 (6·5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1. Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E. coli.

The authors have characterized a chromosomally localized two-gene operon, cueAR, which encodes a putative P1-type ATPase, CueA, and a MerR-type metalloregulatory protein, CueR, in Pseudomonas putida PNL-MK25. Disruption of cueAR by the insertion of mini-Tn5::gfp into the wild-type strain led to a mutant strain with a sixfold reduction in its tolerance to copper; however, the tolerance of this mutant strain to the other seven related transition metals tested was not affected. The sensitivity of the mutant strain was attributed to a higher level of accumulation of intracellular copper, suggesting the involvement of CueA in copper export. Insertion of the cloned cueAR operon into the copper-sensitive mutant strain fully restored its tolerance to copper. cueA::gfp expression studies confirmed that the cueAR operon was transcriptionally regulated by copper and CueR. Studies done on the mutant strain complemented with cueR and cueA revealed partial functional redundancy of cueA and cueR, respectively, in copper tolerance. Thus, the results of this study clearly suggest the involvement of cueAR in copper homeostasis in P. putida.