Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum

Non-mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor-growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino-terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA(-) cells was associated with the impairment of phagocytosis and fluid-phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.     

Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii

The C-terminal coding region of the gene (denoted cshA) encoding a high-molecular-mass (290 kDa) cell-surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3' end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell-surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus-anchored cell-surface protein determinant of hydrophobicity and coaggregation in S. gordonii.     

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.     

rpoS基因在肠杆菌CGMCC 5087响应环境胁迫中的功能

【背景】苯乙醇(2-Phenylethanol,2-PE)是一种具有玫瑰香气味的高级香料添加剂,被广泛应用于香水、化妆品、食品和医药等领域。目前,利用工程菌合成苯乙醇有很好的应用前景。我们分离到一株肠杆菌(Enterobacter sp.) CGMCC 5087,其可以通过苯丙酮酸途径合成2-PE。然而该菌的生长受到不同环境因素导致的胁迫,进而影响苯乙醇的产量。RpoS作为一种稳定期σ因子和压力应答过程中的主要调节因子,在细菌抗环境胁迫生长中起重要作用。【目的】阐明肠杆菌CGMCC 5087中rpoS基因在多种环境胁迫中的作用,掌握该菌在不同环境胁迫下的生物学特性。【方法】使用CRISPR基因编辑技术敲除rpoS基因,通过质粒表达系统构建互补菌株,检测rpoS基因缺失株ΔrpoS与野生型WT菌株和互补菌株ΔrpoS(rpoS)在高渗透压、高温、低pH和氧化应激环境下的生长情况,并进行统计学分析。【结果】rpoS基因的缺失显著降低了肠杆菌CGMCC 5087的生长。在5%NaCl和pH 5.0胁迫条件下,rpoS基因的缺失导致肠杆菌CGMCC 5087的耐受性显著降低。在42℃高温条件下,rpoS基因的缺失导致肠杆菌CGMCC 5087在对数期的耐受性显著降低,而在衰退期的耐受性增强。1 mmol/L H2O2氧化胁迫条件下,rpoS基因的缺失导致肠杆菌CGMCC 5087的延滞期延长,而进入稳定期后rpoS基因突变株耐受性较野生型菌株明显增强。【结论】在肠杆菌CGMCC 5087中,RpoS在抵抗多种环境压力中均具有重要作用,而且在菌株不同的生长时期对于环境胁迫的应答也有所不同,为进一步了解肠杆菌CGMCC 5087的生物学特性、掌握RpoS在肠杆菌CGMCC 5087合成苯乙醇过程中的作用机制提供基础。     

Combined stress effect of pH and temperature narrows the niche width of flagellates in acid mining lakes

Strains of the green alga Chlamydomonas acidophila and two chrysomonads, Ochromonas spp., isolated from each of two similar acid mining lakes (AMLs) with extremely low pH (～2.6) were investigated to consider a possible synergistic stress effect of low pH and unfavourable temperature. We measured flagellate growth rates over a combination of four pH (2.5, 3.5, 5.0 and 7.0) and three temperatures (10, 17.5 and 25°C) in the laboratory. Our hypothesis was that, under highly acidic conditions (pH <3), an obligate acidophil species (C. acidophila) would be less sensitive to the combined stress of pH and temperature than acidotolerant species (Ochromonas spp.). We expected that the difference of the fundamental vs. realized pH niche would be greater in the latter. Another chrysomonad, Poterioochromonas malhamensis strain DS, served as a reference for a closely related neutrophil species. Surprisingly, C. acidophila did not survive temperatures >27°C. The lowest temperature tested reduced growth rates of all three chrysomonad strains significantly. Since all chrysomonads were tolerant to high temperature, growth rate of one Ochromonas spp. strain was measured exemplarily at 35°C. Only at this high temperature was the realized pH niche significantly narrowed. We also recorded significant intraspecific differences within the C. acidophila strains from the two AML, illustrating that the niche width of a species is broader than that of individual clones.     

The fungal type II myosin in Penicillium marneffei, MyoB, is essential for chitin deposition at nascent septation sites but not actin localization

Cytokinesis is essential for proliferative growth but also plays equally important roles during morphogenesis and development. The human pathogen Penicillium marneffei is capable of dimorphic switching in response to temperature, growing in a multicellular filamentous hyphal form at 25°C and in a unicellular yeast form at 37°C. P. marneffei also undergoes asexual development at 25°C to produce multicellular differentiated conidiophores. Thus, P. marneffei exhibits cell division with and without cytokinesis and division by budding and fission, depending on the cell type. The type II myosin gene, myoB, from P. marneffei plays important roles in the morphogenesis of these cell types. Deletion of myoB leads to chitin deposition defects at sites of cell division without perturbing actin localization. In addition to aberrant hyphal cells, distinct conidiophore cell types are lacking due to malformed septa and nuclear division defects. At 37°C, deletion of myoB prevents uninucleate yeast cell formation, instead producing long filaments resembling hyphae at 25°C. The ΔmyoB cells also often lyse due to defects in cell wall biogenesis. Thus, MyoB is essential for correct morphogenesis of all cell types regardless of division mode (budding or fission) and defines differences between the different types of growth.     

Cell Wall-Anchored CshA Polypeptide (259 Kilodaltons) in Streptococcus gordonii Forms Surface Fibrils That Confer Hydrophobic and Adhesive Properties

It has been shown previously that inactivation of the cshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 +/- 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of cshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire cshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous Enterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over E. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in E. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stained E. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 +/- 9.1 nm long that were absent from control E. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordonii adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral "sanguis-like" streptococci.     

Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilus

Pseudouridine at position 55 (Ψ55) in eubacterial tRNA is produced by TruB. To clarify the role of the Ψ55 modification, we constructed a truB gene disruptant (ΔtruB) strain of Thermus thermophilus which is an extreme-thermophilic eubacterium. Unexpectedly, the ΔtruB strain exhibited severe growth retardation at 50 °C. We assumed that these phenomena might be caused by lack of RNA chaperone activity of TruB, which was previously hypothetically proposed by others. To confirm this idea, we replaced the truB gene in the genome with mutant genes, which express TruB proteins with very weak or no enzymatic activity. However the growth retardation at 50 °C was not rescued by these mutant proteins. Nucleoside analysis revealed that Gm18, m(5)s(2)U54 and m(1)A58 in tRNA from the ΔtruB strain were abnormally increased. An in vitro assay using purified tRNA modification enzymes demonstrated that the Ψ55 modification has a negative effect on Gm18 formation by TrmH. These experimental results show that the Ψ55 modification is required for low-temperature adaptation to control other modified. (35)S-Met incorporation analysis showed that the protein synthesis activity of the ΔtruB strain was inferior to that of the wild-type strain and that the cold-shock proteins were absence in the ΔtruB cells at 50°C.     

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis

The complex I subunits NuoL, NuoM and NuoN are homologous to two proteins, MrpA and MrpD, from one particular class of Na+/H+ antiporters. In many bacteria MrpA and MrpD are encoded by an operon comprising 6-7 conserved genes. In complex I these protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Deletion of either mrpA or mrpD from the Bacillus subtilis chromosome resulted in a Na+ and pH sensitive growth phenotype. The deletion strains could be complemented in trans by their respective Mrp protein, but expression of MrpA in the B. subtilis ΔmrpD strain and vice versa did not improve growth at pH 7.4. This corroborates that the two proteins have unique specific functions. Under the same conditions NuoL could rescue B. subtilis ΔmrpA, but improved the growth of B. subtilis ΔmrpD only slightly. NuoN could restore the wild type properties of B. subtilis ΔmrpD, but had no effect on the ΔmrpA strain. Expression of NuoM did not result in any growth improvement under these conditions. This reveals that the complex I subunits NuoL, NuoM and NuoN also demonstrate functional specializations. The simplest explanation that accounts for all previous and current observations is that the five homologous proteins are single ion transporters. Presumably, MrpA transports Na+ whereas MrpD transports H+ in opposite directions, resulting in antiporter activity. This hypothesis has implications for the complex I functional mechanism, suggesting that one Na+ channel, NuoL, and two H+ channels, NuoM and NuoN, are present.     

The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex

In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram‐positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein–protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA‐like domains that are found in all DEAD box RNA helicases and a C‐terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C‐terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome.     

The function of micF RNA. micF RNA is a major factor in the thermal regulation of OmpF protein in Escherichia coli

The role of chromosomally derived micF RNA as a repressor of outer membrane protein OmpF of Escherichia coli was examined for various growth conditions. Levels of micF RNA as determined by Northern analyses are found to increase in response to cell growth at high temperature, in high osmolarity or in the presence of ethanol. After a switch to higher growth temperature, the levels of ompF mRNA and of newly synthesized OmpF decrease with time in E. coli strain, MC4100 but these decreases are not observed in isogenic micF deletion strain, SM3001. In addition, while levels of ompF mRNA are substantially reduced in both strains in response to high osmolarity or ethanol at 24 degrees C, the reduced levels in the parental strain are still 4-5-fold lower compared with the micF deletion strain. These findings indicate that chromosomally derived micF RNA plays a major role in the thermal regulation of OmpF and represses OmpF synthesis in response to several environmental signals by decreasing the levels of ompF mRNA. Analyses of the effect of a multicopy micF plasmid on the levels of OmpF and ompF mRNA after an increase in temperature indicated that multicopies of micF RNA markedly inhibited OmpF synthesis but did not accentuate ompF mRNA decrease. These data suggest that multicopy micF inhibits OmpF synthesis primarily through translational inactivation of ompF mRNA and that a limiting factor in addition to micF RNA is necessary to destabilize ompF mRNA.     

A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.     

Complete auxotrophy for unsaturated fatty acids requires deletion of two sets of genes in Mycobacterium smegmatis

The synthesis of unsaturated fatty acids in Mycobacterium smegmatis is poorly characterized. Bioinformatic analysis revealed four putative fatty acid desaturases in its genome, one of which, MSMEG_1886, is highly homologous to desA3, the only palmitoyl/stearoyl desaturase present in the Mycobacterium tuberculosis genome. A MSMEG_1886 deletion mutant was partially auxotrophic for oleic acid and viable at 37°C and 25°C, although with a long lag phase in liquid medium. Fatty acid analysis suggested that MSMEG_1886 is a palmitoyl/stearoyl desaturase, as the synthesis of palmitoleic acid was abrogated, while oleic acid contents dropped by half in the mutant. Deletion of the operon MSMEG_1741‐1743 (highly homologous to a Pseudomonas aeruginosa acyl‐CoA desaturase) had little effect on growth of the parental strain; however the double mutant MSMEG_1886‐MSMEG_1741‐1743 strictly required oleic acid for growth. The ΔMSMEG_1886‐ΔMSMEG_1741 double mutant was able to grow (poorly but better than the ΔMSMEG_1886 single mutant) in solid and liquid media devoid of oleic acid, suggesting a repressor role for ΔMSMEG_1741. Fatty acid analysis of the described mutants suggested that MSMEG_1742‐43 desaturates C18:0 and C24:0 fatty acids. Thus, although the M. smegmatis desA3 homologue is the major player in unsaturated fatty acid synthesis, a second set of genes is also involved.     

Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus

Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.