Null mutations in the essential gene yrfF (mucM) are not lethal in rcsB,yojN or rcsC strains of Salmonella enterica serovar Typhimurium

Insertion of factor MudJ in the intergenic region between divergent genes yrfF and yrfE, at centisome 76 in the genome of Salmonella enterica serovar Typhimurium LT2, confers the characteristics recently described for mucM mutants, i.e. mucoidy and resistance to mecillinam. Cloning of the intergenic region plus either the yrfF or the yrfE gene in a multicopy plasmid showed that only the plasmid carrying the yrfF gene complemented mucM mutants, thus suggesting that mucM mutations are in fact yrfF mutations. A null yrfF mutation obtained by insertion of a kanamycin cassette into the yrfF open reading frame (yrfF28::Kan) produced abortive colonies when transduced to a wild-type strain but was normally accepted by rcsB, rcsC or yojN strains. Neither mutations preventing synthesis of the capsular exopolysaccharide colanic acid (cps, galE) nor rcsA mutations, which reduce expression of cps genes, conferred tolerance to the lethal yrfF28::Kan mutation. Spontaneous suppressor mutations arose very frequently in abortive yrfF28::Kan colonies, and all of them affected either rcsC, yojN, or rcsB genes. Thus, the lethal effect caused by inactivation of gene yrfF appears to be mediated by a function that is dependent on the rcsC-yojN-rcsB phosphorelay system but does not involve synthesis of colanic acid.     

Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.     

Penicillin binding protein 5 affects cell diameter, contour, and morphology of Escherichia coli

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of Escherichia coli, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of E. coli mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in DeltadacA mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.     

Compensatory evolution of pbp mutations restores the fitness cost imposed by β-lactam resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.     

Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants.

Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division.     

Selected amplification of the cell division genes ftsQ-ftsA-ftsZ in Escherichia coli

Rapidly growing Escherichia coli is unable to divide in the presence of the antibiotic mecillinam, whose direct target is penicillin-binding protein 2 (PBP2), responsible for the elongation of the cylindrical portion of the cell wall. Division can be restored in the absence of PBP2 activity by increasing the concentration of the cell division proteins FtsQ, FtsA, and FtsZ. We tried to identify regulators of the ftsQ-ftsA-ftsZ operon among mecillinam-resistant mutants, which include strains overexpressing these genes. By insertional mutagenesis with mini-Tn10 elements, we selected for insertions that conferred mecillinam resistance. Among 15 such mutants, 7 suppressed the thermosensitivity of the ftsZ84(Ts) mutant, strongly suggesting that they had increased FtsZ activity. In all 7 cases, however, the mutants resulted from a duplication of the ftsQAZ region. These duplications seemed to result from multiple events, suggesting that no simple insertional inactivation can result in a mutant with sufficiently amplified ftsQAZ expression to confer mecillinam resistance. The structure of the duplications suggests a general method for constructing directed duplications of precise sequences.     

Enterococcus faecalis multi-drug resistance transporters: application for antibiotic discovery

Using bioinformatics approaches, 34 potential multidrug resistance (MDR) transporter sequences representing 4 different transporter families were identified in the unannotated Enterococcus faecalis database (TIGR). A functional genomics campaign generating single-gene insertional disruptions revealed several genes whose absence confers significant hypersensitivities to known antimicrobials. We constructed specific strains, disrupted in a variety of previously unpublished, putative MDR transporter genes, as tools to improve the success of whole-cell antimicrobial screening and discovery. Each of the potential transporters was inactivated at the gene level and then phenotypically characterized, both with single disruption mutants and with 2-gene mutants built upon a delta norA deleted strain background.     

Differential effect of mutational impairment of penicillin-binding proteins 1A and 1B on Escherichia coli strains harboring thermosensitive mutations in the cell division genes ftsA, ftsQ, ftsZ, and pbpB.

To study the functional differences between penicillin-binding proteins (PBPs) 1A and 1B, as well as their recently postulated involvement in the septation process (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989), a series of isogenic strains with mutations in the genes coding for PBP 1A (ponA) or PBP 1B (ponB) or in the cell division-specific genes ftsA, ftsQ, pbpB, and ftsZ was constructed and used as the start point to produce double mutants combining the ponA or ponB characters with mutations in cell division genes. PBP 1A seemed to be unable to preserve cell integrity by itself, requiring the additional activities of PBP 2, PBP 3, and FtsQ. PBP 1B was apparently endowed with a more versatile biosynthetic potential that permitted a substantial enlargement of PBP 1A-deficient cells when PBP 2 or 3 was inhibited or when FtsQ was inactive. beta-Lactams binding to PBP 2 (mecillinam) or 3 (furazlocillin) caused rapid lysis in a ponB background. The lytic effect of furazlocillin to ponB cell division double mutants was suppressed at the restrictive temperature irrespective of the identity of the mutated cell division gene. These results indicate that PBPs 1A and 1B play distinct roles in cell wall synthesis and support the idea of a relevant involvement of PBP 1B in peptidoglycan synthesis at the time of septation.     

Genome-wide identification of ampicillin resistance determinants in Enterococcus faecium

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism.     

Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae.

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.     

Penicillin-binding proteins in β-lactam-resistant laboratory mutants of Streptococcus pneumoniae

The increasing number of penicillin-resistant clinical strains of Strepfococcus pneumoniae has raised questions about the mechanism involved. We have isolated a large number of independent, spontaneous laboratory mutants with increasing resistance against either piperacillin or cefotaxime. Both classes of mutants showed a different pathway of penicillin-binding protein (PBP) alterations, and within each group of mutants the individual PBPs appeared to have changed at different resistance levels and in different sequences. The mutations led to decreased β-lactam affinity and possibly to a reduction in the amount of protein present in the cell, but differences in apparent molecular weight, like those reported in low- and high-level resistant pathogenic strains, were not found. Some mutants showed a high degree of cross-resistance to a variety of pencillins and cephaiosporins independently of the acquired PBP alterations, indicating that different genotypes can be responsible for the same phenotypic expression of resistance.     

Penicillin-binding proteins in beta-lactam-resistant laboratory mutants of Streptococcus pneumoniae

The increasing number of penicillin-resistant clinical strains of Streptococcus pneumoniae has raised questions about the mechanism involved. We have isolated a large number of independent, spontaneous laboratory mutants with increasing resistance against either piperacillin or cefotaxime. Both classes of mutants showed a different pathway of penicillin-binding protein (PBP) alterations, and within each group of mutants the individual PBPs appeared to have changed at different resistance levels and in different sequences. The mutations led to decreased beta-lactam affinity and possibly to a reduction in the amount of protein present in the cell, but differences in apparent molecular weight, like those reported in low- and high-level resistant pathogenic strains, were not found. Some mutants showed a high degree of cross-resistance to a variety of penicillins and cephalosporins independently of the acquired PBP alterations, indicating that different genotypes can be responsible for the same phenotypic expression of resistance.     

Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.     

Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.     

Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants.

In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.     

Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin.

Penicillin-binding protein (PBP) 5 of Streptococcus faecium ATCC 9790 has an unusually low affinity for penicillin (50% binding occurred at a penicillin level of 8 micrograms/ml after 60 min of incubation, and the protein only became labeled after 20 min of incubation with high concentrations of radioactive penicillin). PBPs with similar properties are carried by strains of Streptococcus durans, Streptococcus faecalis, and Streptococcus lactis but not by strains of groups A, B, C, and G streptococci or Streptococcus pneumoniae. The strains carrying the slow-reacting PBP demonstrated a sensitivity to penicillin that was several hundred times lower than that of strains not carrying it. Spontaneous mutants with minimal inhibitory concentrations of penicillin of 20, 40, and 80 micrograms/ml were isolated from S. faecium ATCC 9790. They all showed a dramatic increase in the amount of slow-reacting PBP produced. Mutants with increased penicillin resistance were also isolated from wild-type strains of S. durans, S. faecalis, and S. faecium. All of them carried a greater amount of the slow-reacting PBP than that carried by the parent. Finally, it was found that resistant S. faecium ATCC 9790 mutants grew normally in the presence of penicillin concentrations that were far above that saturating all PBPs except PBP 5. Cell growth was, on the contrary, inhibited by a penicillin concentration that saturated the slow-reacting PBP by 90%. This penicillin dose was equal to the minimal inhibitory concentration.     

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli

Escherichia coli has 12 recognized penicillin binding proteins (PBPs), four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase activity. Although the enzymology of the DD-carboxypeptidases has been studied extensively, the in vivo functions of these proteins are poorly understood. To explain why E. coli maintains four independent loci encoding enzymes of considerable sequence identity and comparable in vitro activity, it has been proposed that the DD-carboxypeptidases may substitute for one another in vivo. We tested the validity of this equivalent substitution hypothesis by investigating the effects of these proteins on the aberrant morphology of DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.     

在全基因组范围内筛选酵母中砷抗性相关基因

砷化物是广泛应用的抗癌药物,特别是对白血病有显著疗效.然而,治疗过程中病人会因对砷化物具有耐药性而影响治疗效果,而目前对于砷抗性机制尚缺全面深入的研究.利用酵母作为模式生物,使用不同浓度的砷对由4757个酵母缺失型突变体组成的菌株库进行筛查.共鉴定出104个基因/开放阅读框(ORF),其缺失导致酵母对砷的抗性增加.生物信息学分析结果提示,这些基因与mRNA分解代谢、应激反应、组蛋白乙酰化和蛋白质合成及分解代谢等功能有关.同时这些基因中多于半数具有哺乳动物同源类似物.所以,进一步研究这些基因有望为人类砷化物的耐药性及毒性机制研究提供富有价值的新线索.