Prevalence and Antifungal Susceptibility of Candida parapsilosis Species Complex in Eastern China: A 15-Year Retrospective Study by ECIFIG

Candida parapsilosis complex is one of the most common non-albicans Candida species that cause candidemia, especially invasive candidiasis. The purpose of this study was to evaluate the antifungal susceptibilities of both colonized and invasive clinical C. parapsilosis complex isolates to 10 drugs: amphotericin (AMB), anidulafungin (AFG), caspofungin (CAS), micafungin (MFG), fluconazole (FLZ), voriconazole (VRZ), itraconazole (ITZ), posaconazole (POZ), 5-flucytosine (FCY), and isaconazole (ISA). In total, 884 C. parapsilosis species complex isolates were gathered between January 2005 and December 2020. C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis accounted for 86.3, 8.1, and 5.5% of the cryptic species, respectively. The resistance/non-wild-type rate of bloodstream C. parapsilosis to the drugs was 3.5%, of C. metapsilosis to AFG and CAS was 7.7%, and of C. orthopsilosis to FLZ and VRZ was 15% and to CAS, MFG, and POZ was 5%. The geometric mean (GM) minimum inhibitory concentrations (MICs) of non-bloodstream C. parapsilosis for CAS (0.555 mg/L), MFG (0.853 mg/L), FLZ (0.816 mg/L), VRZ (0.017 mg/L), ITZ (0.076 mg/L), and POZ (0.042 mg/L) were significantly higher than those of bloodstream C. parapsilosis, for which the GM MICs were 0.464, 0.745, 0.704, 0.015, 0.061, and 0.033 mg/L, respectively (P < 0.05). The MIC distribution of the bloodstream C. parapsilosis strains collected from 2019 to 2020 for VRZ, POZ, and ITZ were 0.018, 0.040, and 0.073 mg/L, significantly higher than those from 2005 to 2018, which were 0.013, 0.028, and 0.052 mg/L (P < 0.05). Additionally, MIC distributions of C. parapsilosis with FLZ and the distributions of C. orthopsilosis with ITZ and POZ might be higher than those in Clinical and Laboratory Standards Institute studies. Furthermore, a total of 143 C. parapsilosis complex isolates showed great susceptibility to ISA. Overall, antifungal treatment of the non-bloodstream C. parapsilosis complex isolates should be managed and improved. The clinicians are suggested to pay more attention on azoles usage for the C. parapsilosis complex isolates. In addition, establishing the epidemiological cutoff values (ECVs) for azoles used in Eastern China may offer better guidance for clinical treatments. Although ISA acts on the same target as other azoles, it may be used as an alternative therapy for cases caused by FLZ- or VRZ-resistant C. parapsilosis complex strains.

Functional Redundancy in the Hydroxycinnamate Catabolism Pathways of the Salt Marsh BacteriumSagittula stellataE-37

ABSTRACTThe hydroxycinnamates (HCAs) ferulate andp-coumarate are among the most abundant constituents of lignin, and their degradation by bacteria is an essential step in the remineralization of vascular plant material. Here, we investigate the catabolism of these two HCAs by the marine bacteriumSagittula stellataE-37, a member of the roseobacter lineage with lignolytic potential. Bacterial degradation of HCAs is often initiated by the activity of a hydroxycinnamoyl-coenzyme A (hydroxycinnamoyl-CoA) synthase. Genome analysis ofS. stellatarevealed the presence of two feruloyl-CoA (fcs) synthase homologs, an unusual occurrence among characterized HCA degraders. In order to elucidate the role of these homologs in HCA catabolism,fcs-1andfcs-2were disrupted using insertional mutagenesis, yielding both single and doublefcsmutants. Growth onp-coumarate was abolished in thefcsdouble mutant, whereas maximum cell yield on ferulate was only 2% of that of the wild type. Interestingly, the single mutants demonstrated opposing phenotypes, where thefcs-1mutant showed impaired growth (extended lag and ∼60% of wild-type rate) onp-coumarate, and thefcs-2mutant showed impaired growth (extended lag and ∼20% of wild-type rate) on ferulate, pointing to distinct but overlapping roles of the encodedfcshomologs, withfcs-1primarily dedicated top-coumarate utilization andfcs-2playing a dominant role in ferulate utilization. Finally, a tripartite ATP-independent periplasmic (TRAP) family transporter was found to be required for growth on both HCAs. These findings provide evidence for functional redundancy in the degradation of HCAs inS. stellataE-37 and offer important insight into the genetic complexity of aromatic compound degradation in bacteria.IMPORTANCEHydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacteriumSagittula stellata. We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude thatS. stellatais uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors.

Multiplicity of Sulfate and Molybdate Transporters and Their Role in Nitrogen Fixation in Rhizobium leguminosarum bv. viciae Rlv3841

Rhizobium leguminosarum Rlv3841 contains at least three sulfate transporters, i.e., SulABCD, SulP1 and SulP2, and a single molybdate transporter, ModABC. SulABCD is a high-affinity transporter whose mutation prevented growth on a limiting sulfate concentration, while SulP1 and SulP2 appear to be low-affinity sulfate transporters. ModABC is the sole high-affinity molybdate transport system and is essential for growth with NO3− as a nitrogen source on limiting levels of molybdate (<0.25 μM). However, at 2.5 μM molybdate, a quadruple mutant with all four transporters inactivated, had the longest lag phase on NO3−, suggesting these systems all make some contribution to molybdate transport. Growth of Rlv3841 on limiting levels of sulfate increased sulB, sulP1, modB, and sulP2 expression 313.3-, 114.7-, 6.2-, and 4.0-fold, respectively, while molybdate starvation increased only modB expression (three- to 7.5-fold). When grown in high-sulfate but not low-sulfate medium, pea plants inoculated with LMB695 (modB) reduced acetylene at only 14% of the wild-type rate, and this was not further reduced in the quadruple mutant. Overall, while modB is crucial to nitrogen fixation at limiting molybdate levels in the presence of sulfate, there is an unidentified molybdate transporter also capable of sulfate transport. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY Attribution 4.0 International license .

White Mutants of Chloroperoxidase-Secreting Caldariomyces fumago as Superior Production Strains, Revealing an Interaction between Pigmentation and Enzyme Secretion

ABSTRACTBy mutant-colony screening ofCaldariomyces fumago, several white mutants were isolated that are superior strains for the production of the valuable enzyme chloroperoxidase (CPO). Their culture supernatant lacks the contaminating dark pigment, which simplifies downstream processing. Furthermore, the CPO content increased significantly faster than the wild-type rate, which uncovers possible interactions between pigmentation and enzyme secretion.

Pyruvate Is Synthesized by Two Pathways in Pea Bacteroids with Different Efficiencies for Nitrogen Fixation

ABSTRACT Nitrogen fixation in legume bacteroids is energized by the metabolism of dicarboxylic acids, which requires their oxidation to both oxaloacetate and pyruvate. In alfalfa bacteroids, production of pyruvate requires NAD+ malic enzyme (Dme) but not NADP+ malic enzyme (Tme). However, we show that Rhizobium leguminosarum has two pathways for pyruvate formation from dicarboxylates catalyzed by Dme and by the combined activities of phosphoenolpyruvate (PEP) carboxykinase (PckA) and pyruvate kinase (PykA). Both pathways enable N2 fixation, but the PckA/PykA pathway supports N2 fixation at only 60% of that for Dme. Double mutants of dme and pckA/pykA did not fix N2. Furthermore, dme pykA double mutants did not grow on dicarboxylates, showing that they are the only pathways for the production of pyruvate from dicarboxylates normally expressed. PckA is not expressed in alfalfa bacteroids, resulting in an obligate requirement for Dme for pyruvate formation and N2 fixation. When PckA was expressed from a constitutive nptII promoter in alfalfa dme bacteroids, acetylene was reduced at 30% of the wild-type rate, although this level was insufficient to prevent nitrogen starvation. Dme has N-terminal, malic enzyme (Me), and C-terminal phosphotransacetylase (Pta) domains. Deleting the Pta domain increased the peak acetylene reduction rate in 4-week-old pea plants to 140 to 150% of the wild-type rate, and this was accompanied by increased nodule mass. Plants infected with Pta deletion mutants did not have increased dry weight, demonstrating that there is not a sustained change in nitrogen fixation throughout growth. This indicates a complex relationship between pyruvate synthesis in bacteroids, nitrogen fixation, and plant growth.

The Pseudomonas aeruginosa devB/SOL Homolog,pgl, Is a Member of the hex Regulon and Encodes 6-Phosphogluconolactonase

ABSTRACT A cyclic version of the Entner-Doudoroff pathway is used byPseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form thehex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOLfamily of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5′ end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgland propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

ABSTRACT Two different Cd2+ uptake systems were identified inLactobacillus plantarum. One is a high-affinity, high-velocity Mn2+ uptake system which also takes up Cd2+ and is induced by Mn2+ starvation. The calculated Km and V maxare 0.26 μM and 3.6 μmol g of dry cell−1min−1, respectively. Unlike Mn2+ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd2+ uptake is weakly inhibited by citrate. Cd2+ and Mn2+ are competitive inhibitors of each other, and the affinity of the system for Cd2+ is higher than that for Mn2+. The other Cd2+uptake system is expressed in Mn2+-sufficient cells, and noKm can be calculated for it because uptake is nonsaturable. Mn2+ does not compete for transport through this system, nor does any other tested cation, i.e., Zn2+, Cu2+, Co2+, Mg2+, Ca2+, Fe2+, or Ni2+. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn2+-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn2+ for growth as the parental strain. Mn2+ starvation-induced Cd2+ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn2+ or Cd2+accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn2+ and Cd2+uptake system.

A ubiquitin mutant with specific defects in DNA repair and multiubiquitination.

The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitin-conjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.

ESCAPE FROM MATING-TYPE INCOMPATIBILITY IN BISEXUAL (A + a) NEUROSPORA HETEROKARYONS

In Neurospora crassa, strains of opposite mating type generally do not form stable heterokaryons because the mating type locus acts as a heterokaryon incompatibility locus. However, when one A and one a strain, having complementing auxotrophic mutants, are placed together on minimal medium, growth may occur, although the growth is generally slow. In this study, escape from such slow growth to that at a wild type or near-wild type rate was observed. The escaped cultures are stable heterokaryons, mostly having lost the mating type allele function from one component nucleus, so that the nuclear types are heterokaryon compatible. Either A or a mating type can be lost. This loss of function has been attributed to deletion since only one nuclear type could be recovered in all heterokaryons except one, but deletion spanning adjacent loci has been directly demonstrated in a minority of cases. Alternatively when one component strain is tol and the other tol+ (tol being a recessive mutant suppressing the heterokaryon incompatibility associated with mating type), escape may occur by the deletion or mutation of tol+, also resulting in heterokaryon compatibility. An induction mechanism for escape is speculated upon.