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Dimorphic species of Mucor, which are cosmopolitan fungi belonging to subphylum Mucoromycotina, are metabolically versatile. Some species of Mucor are sources of biotechnological products, such as biodiesel from Mucor circinelloides and expression of heterologous proteins from Mucor lusitanicus. Furthermore, Mucor lusitanicus has been described as a model for understanding mucormycosis infections. However, little is known regarding the relationship between Mucor lusitanicus and other soil inhabitants. In this study, we investigated the potential use of Mucor lusitanicus as a biocontrol agent against fungal phytopathogens, namely Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and Alternaria solani, which destroy economically important crops. Results showed that aerobic cell-free supernatants of the culture broth (SS) from Mucor lusitanicus inhibited the growth of the fungal phytopathogens in culture, soil, and tomato fruits. The SS obtained from a strain of Mucor lusitanicus carrying the deletion of rfs gene, which encodes an enzyme involved in the synthesis of siderophore rhizoferrin, had a decreased inhibitory effect against the growth of the phytopathogens. Contrarily, this inhibitory effect was more evident with the SS from an rfs-overexpressing strain compared to the wild-type. This study provides a framework for the potential biotechnological use of the molecules secreted from Mucor lusitanicus in the biocontrol of fungal phytopathogens.

Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1(+)(−) and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1(+)(−) and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose. To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.

The geothermal zone of Araró, México, is located within the trans-Mexican volcanic belt, an area with numerous arsenic (As)-rich hot springs. In this study, the draft genome sequence of two endemic Bacillus strains (ZAP17 and ZAP62) from Araró microbial mat hot springs was determined, which were able to grow on arsenate (up to 64 mM) and arsenite (up to 32 mM). Phylogenetic analysis based on 16S rRNA and gyrB sequences, as well as genome sequence analysis based on average nucleotide identity (>96%) and digital DNA–DNA hybridization (>70%), indicated that these strains belong to the Bacillus paralicheniformis ZAP17 and Bacillus altitudinis ZAP62. Furthermore, through genome mining, it was identified two arsenic resistance operons, arsRBC, and arsRBCDA in both strains as potential determinants of arsenic (As) resistance. Predicted ArsA (arsenial pump-driving ATPase), ArsB (Arsenical efflux pump protein), ArsC (Arsenate reductase), ArsD (Arsenical efflux pump protein) and ArsR (Metalloregulator/ars operon repressor) proteins, clearly grouped with their respective clades corresponding to other characterized bacterial species, mainly Firmicutes. To further evaluate the functionality of the ars operons in ZAP17 and ZAP62 strains, our results showed that arsRBC and arsRBCDA genes were expressed in the presence of arsenite (III). Finally, the presence of ars operons in the genome of Bacillus species residing in As-rich environments, such as the Araró hot springs, might be a potential mechanism to survive under such harsh conditions, as well as to design sustainable bioremediation strategies.

Mucor circinelloides is a fungus that produces diverse spores throughout its life cycle. The sporangiospores, which are the most well-studied spores in this fungus, are asexual spores produced during aerial mycelial development. M. circinelloides has the potential to be used in diverse biotechnological applications. In this study, we propose rice (Oryza sativa) grains as an alternative substrate for inexpensive and large-scale sporangiospore production. The sporangiospores produced from rice and a yeast extract-peptone-glucose (YPG) medium exhibited similar protein and nucleic acid contents and phenotypes in terms of germination under different conditions and culture media, including similar virulence rates against the nematode Caenorhabditis elegans. Transgenic strains carrying self-replicative plasmids were sporulated on rice and showed plasmid stability similar to that of spores produced on the YPG medium. Approximately 20% of the spore population lost plasmids after the first passage on rice. These results reveal that rice is a suitable substrate for the mass production of sporangiospores in M. circinelloides. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02853-1.

Fusarium oxysporum f. sp. lycopersici is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In F. oxysporum many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in F. oxysporum f. sp. lycopersici, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each arf encoding gene revealed that the highest expression corresponds to arf1 in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in F. oxysporum f. sp. lycopersici. The essential function suggested of Arf1 in F. oxysporum f. sp. lycopersici was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in arf1. Moreover, arl3 mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆arl3 was less virulent compared to the wild-type strain. These results describe the role of arl3 as a critical regulator of the virulence in F. oxysporum f. sp. lycopersici and stablish a framework for the arf family members to be studied in deeper details in this phytopathogen.

Mucor circinelloides, a dimorphic opportunistic pathogen, expresses three heterotrimeric G-protein beta subunits (Gpb1, Gpb2 and Gpb3). The Gpb1-encoding gene is up-regulated during mycelial growth compared with that in the spore or yeast stage. gpb1 deletion mutation analysis revealed its relevance for an adequate development during the dimorphic transition and for hyphal growth under low oxygen concentrations. Infection assays in mice indicated a phenotype with considerably reduced virulence and tissue invasiveness in the deletion mutants (Δgpb1) and decreased host inflammatory response. This finding could be attributed to the reduced filamentous growth in animal tissues compared with that of the wild-type strain. Mutation in a regulatory subunit of cAMP-dependent protein kinase A (PKA) subunit (PkaR1) resulted in similar phenotypes to Δgpb1. The defects exhibited by the Δgpb1 strain were genetically suppressed by pkaR1 overexpression, indicating that the PKA pathway is controlled by Gpb1 in M. circinelloides. Moreover, during growth under low oxygen levels, cAMP levels were much higher in the Δgpb1 than in the wild-type strain, but similar to those in the ΔpkaR1 strain. These findings reveal that M. circinelloides possesses a signal transduction pathway through which the Gpb1 heterotrimeric G subunit and PkaR1 control mycelial growth in response to low oxygen levels.

Mobile plasmid-encoded elements are DNA segments that are transferred for horizontal gene transfer and that confer adaptive proprieties, as well as virulence and antibiotic and heavy metal resistance to bacteria. The conjugative plasmid pUM505, isolated from a clinical strain of Pseudomonas aeruginosa, possesses a putative 31.292 kb mobile element (denominated Mpe: Mobile plasmid- encoded element) that, in addition to possessing chr genes that confer chromate resistance to Pseudomonas, contains two putative mer operons that could confer mercury resistance. Moreover, the Mpe contains genes related previously with the virulence of both P. aeruginosa and Escherichia coli strains. In this work, we determined that Mpe from pUM505 was able to independently move to another DNA molecule, conferring chromate and mercury resistance to P. aeruginosa PAO1 and mercury resistance to E. coli JM101, suggesting that its transference might be beneficial to bacteria under certain environmental conditions. Additionally, the transference of Mpe increased the virulence of P. aeruginosa PAO1 against the nematode Caenorhabditis elegans, suggesting its contribution to the pathogenicity of P. aeruginosa. In this work, we describe a new mobile plasmid-encoded element that possesses the potential to be transferred by horizontal gene transference, which could provide bacteria with a wide variety of adaptive traits such as heavy metal resistance and virulence, which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.

Mucor circinelloides is a dimorphic fungus used to study cell differentiation that has emerged as a model to characterize mucormycosis. In this work, we identified four ADP-ribosylation factor (Arf)-encoding genes (arf1-arf4) and study their role in the morphogenesis and virulence. Arfs are key regulators of the vesicular trafficking process and are associated with both growth and virulence in fungi. Arf1 and Arf2 share 96% identity and Arf3 and Arf4 share 89% identity, which suggests that the genes arose through gene-duplication events in M. circinelloides. Transcription analysis revealed that certain arf genes are affected by dimorphism of M. circinelloides, such as the arf2 transcript, which was accumulated during yeast development. Therefore, we created knockout mutants of four arf genes to evaluate their function in dimorphism and virulence. We found that both arf1 and arf2 are required for sporulation, but these genes also perform distinct functions; arf2 participates in yeast development, whereas arf1 is involved in aerobic growth. Conversely, arf3 and arf4 play only minor roles during aerobic growth. Moreover, we observed that all single arf-mutant strains are more virulent than the wild-type strain in mouse and nematode models, with the arf3 mutant being most virulent. Lastly, arf1/arf2 and arf3/arf4 double mutations produced heterokaryon strains that did not reach the homokaryotic state, indicating that these genes participate in essential and redundant functions. Overall, this work reveals that Arfs proteins regulate important cellular processes in M. circinelloides such as morphogenesis and virulence, laying the foundation to characterize the molecular networks underlying this regulation.

The ciprofloxacin-resistance crpP gene, encoded by the pUM505 plasmid, isolated from a P. aeruginosa clinical isolate, confers an enzymatic mechanism of antibiotic phosphorylation, which is ATP-dependent, that decreases ciprofloxacin susceptibility. Homologous crpP genes are distributed across extended spectrum beta-lactamase (ESBL)-producing isolates obtained from Mexican hospitals and which confer decreased susceptibility to CIP. The analysis of sequences of the CrpP of proteins showed that the residues Gly7, Thr8, Asp9, Lys33 and Gly34 (located at the N-terminal region) and Cys40 (located at the C-terminal region) are conserved in all proteins, suggesting that these residues could be essential for CrpP function. The aim of this study was to investigate the amino acids essential to ciprofloxacin resistance, which is conferred by the CrpP protein of pUM505 plasmid. Mutations in the codons encoding Gly7, Asp9, Lys33 and Cys40 of CrpP protein from pUM505 were generated by PCR fusion. The results showed that all mutations generated in CrpP proteins increased ciprofloxacin susceptibility in Escherichia coli . In addition, the CrpP modified proteins were purified and their enzymatic activity on ciprofloxacin was assayed, showing that these modified proteins do not exert catalytic activity on ciprofloxacin. Moreover, by infrared assays it was determined that the modified proteins were are not able to modify the ciprofloxacin molecule. Our findings are the first report that indicate that the amino acids, namely Gly7, Asp9, Lys33 and Cys40, which are conserved in the CrpP proteins, possess an essential role for the enzymatic mechanism that confers ciprofloxacin resistance.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

Objectives This study investigated the presence of the crpP gene, which encodes an enzymatic mechanism of antibiotic phosphorylation that decreases ciprofloxacin susceptibility, in ESBL-producing clinical isolates and its effect in transconjugants. Methods A collection of 77 ESBL-producing clinical isolates of Enterobacteriaceae and 68 ESBL-producing transconjugants that had acquired plasmids from clinical isolates from hospitals in Mexico obtained from 1988 to 2012 was employed. The crpP homologue genes were identified by dot-blot and PCR assays; five of them were sequenced and an in silico analysis was conducted. Expression of CrpP proteins was determined by western blot assays using antibodies against CrpP from plasmid pUM505. Three crpP homologue genes were cloned and transferred to Escherichia coli J53-3 as recipient strain. Results The crpP gene was identified in four (5.19%) ESBL-producing isolates and five (7.35%) ESBL-producing transconjugants with plasmids from clinical isolates. Analysis of the deduced amino acid (aa) sequence of the CrpP protein homologues revealed that they all corresponded to small proteins (63-70 aa) with an identity of 10.1%-43.7% with respect to the pUM505 CrpP sequence. In addition, all crpP-positive transconjugants expressed a CrpP protein. Finally, transfer of crpP homologues conferred lower ciprofloxacin susceptibility to E. coli. Conclusions These findings indicate the presence of crpP genes among ESBL-producing isolates from Mexican hospitals and point to widespread crpP-type genes in old Enterobacteriaceae clinical isolates (from 1994). CrpP probably confers resistance by means of the phosphorylation of ciprofloxacin.

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP , for c iprofloxacin r esistance p rotein, p lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC- crpP , conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography–tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa , which involves phosphorylation of the antibiotic.

The pUM505 plasmid was isolated from a clinical strain of Pseudomonas aeruginosa. This plasmid contains a genomic island with sequence similar to islands found in chromosomes of virulent P. aeruginosa clinical isolates. The objective of this work was to determine whether pUM505 increases the virulence of P. aeruginosa and to identify the genes responsible for this property. First, using the lettuce-leaf model, we found that pUM505 significantly increases the virulence of P. aeruginosa reference strain PAO1. pUM505 also increased the PAO1 virulence in a murine model and increased cytotoxicity of this strain toward HeLa cells. Thus, we generated a pUM505 gene library of 103 clones in the pUCP20 binary vector. The library was transferred to Escherichia coli TOP10 and P. aeruginosa PAO1 to identify genes. The lettuce-leaf model allowed us to identify three recombinant plasmids that increased the virulence of both E. coli and P. aeruginosa strains. These recombinant plasmids also increased the virulence of the PAO1 strain in mice and induced a cytotoxic effect in HeLa cells. Eleven genes were identified in the virulent transformants. Of these genes, only the pUM505 ORF 2 has homology with a gene previously implicated in virulence. These results indicate that pUM505 contains several genes that encode virulence factors, suggesting that the plasmid may contribute directly to bacterial virulence.

In fungi, heterotrimeric G proteins are key regulators of biological processes such as mating, virulence, morphology, among others. Mucor circinelloides is a model organism for many biological processes, and its genome contains the largest known repertoire of genes that encode putative heterotrimeric G protein subunits in the fungal kingdom: twelve Gα (McGpa1-12), three Gβ (McGpb1-3), and three Gγ (McGpg1-3). Phylogenetic analysis of fungal Gα showed that they are divided into four distinct groups as reported previously. Fungal Gβ and Gγ are also divided into four phylogenetic groups, and to our understanding this is the first report of a phylogenetic classification for fungal Gβ and Gγ subunits. Almost all genes that encode putative heterotrimeric G subunits in M. circinelloides are differentially expressed during dimorphic growth, except for McGpg1 (Gγ) that showed very low mRNA levels at all developmental stages. Moreover, several of the subunits are expressed in a similar pattern and at the same level, suggesting that they constitute discrete complexes. For example, McGpb3 (Gβ), and McGpg2 (Gγ), are co-expressed during mycelium growth, and McGpa1, McGpb2, and McGpg2, are co-expressed during yeast development. These findings provide the conceptual framework to study the biological role of these genes during M. circinelloides morphogenesis.

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin–Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET- pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans , indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.

Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

Mucor circinelloides is an opportunistic human pathogen that is used to study mucormycosis, a rare but lethal infection in susceptible immunosuppressed patients. However, the virulence characteristics of this pathogen have not been fully elucidated. In this study, sporangiospores (spores) produced on YPG medium supplemented with native blood serum increased the virulence of M. circinelloides compared with spores produced on YPG supplemented with denatured blood serum or on YPG alone. The spores produced from YPG supplemented with native blood serum increased nematode death and led to significant increases in interleukin (IL)-6, IL-1β, macrophage inhibitory protein-2, and tumour necrosis factor α mRNA levels in liver and lung tissues from infected diabetic mice compared with those in tissues from animals infected with spores produced in the presence of YPG supplemented with denatured blood serum or of YPG alone. Moreover, spores produced from cultures supplemented with native blood serum showed increased germination rates and longer hyphae compared with other spores. The spores produced in YPG supplemented with native blood serum also enhanced resistance to stress factors and H2O2 and increased thermotolerance compared with spores produced under other conditions. In addition, spores produced in presence of blood serum increased the ability of the pathogen to survive in the presence of macrophages. Taken together, our results showed that these factors were important features for fungal virulence in humans and suggested that thermolabile components in the blood serum may induce M. circinelloides virulence.

The Pseudomonas aeruginosa plasmid pUM505 contains in a pathogenicity island the dsbA2 gene, which encodes a product with similarity to DsbA protein disulfide isomerases, enzymes that catalyze formation and isomerization of disulfide bonds in protein cysteine residues. Using transcriptional fusions, it was found that dsbA2 gene promoter is activated during the stationary phase, suggesting that DsbA2 protein may be required for adaptive changes that occur during this stage of bacterial growth. Transfer of the pUM505 dsbA2 gene to a cadmium-sensitive P. aeruginosa PAO1-derivative affected in the chromosomal dsbA gene, restored cadmium resistance, suggesting a role of DsbA2 in protecting protein disulfide bonds. PAO1 dsbA2 transformants displayed increased sensitivity to intercalating agent mitomycin C, indicating that DsbA2 functions as a thioredoxin enzyme able to modify and activate toxicity of this compound. These results highlight the adaptive role of the pUM505 plasmid in its P. aeruginosa hosts.

Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.

Chromate-hypersensitive mutants of the Pseudomonas aeruginosa PAO1 strain were isolated using transposon-insertion mutagenesis. Comparison of the nucleotide sequences of the regions interrupted in the mutants with the PAO1 genome revealed that the genes affected in three mutant strains were oprE (ORF PA0291), rmlA (ORF PA5163), and ftsK (ORF PA2615), respectively. A relationship of these genes with chromate tolerance has not been previously reported. No other phenotypic changes were observed in the oprE mutant but its resistance to chromate was not fully restored by expressing the ChrA protein, which extrudes chromate ions from the cytoplasm to the periplasmic space. These data suggest that OprE participates in the efflux of chromate from the periplasm to the outside. Increased susceptibility of the rmlA mutant to the metals cadmium and mercury and to the anion-superoxide generator paraquat suggests a protective role of LPS against chromate toxicity. A higher susceptibility of the ftsK mutant to compounds affecting DNA structure (ciprofloxacin, tellurite, mitomycin C) suggests a role of FtsK in the recombinational repair of DNA damage caused by chromate. In conclusion, the P. aeruginosa genome contains diverse genes related to its intrinsic resistance to chromate. Systems pertaining to the outer membrane (OprE), the cell wall (LPS), and the cytoplasm (FtsK) were identified in this work as involved in chromate protection mechanisms.

The chromate ion transporter (CHR) superfamily includes proteins that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 encodes six CHR homologues in its multireplicon genome and has been reported as highly chromate-resistant. The objective of this work was to analyze the involvement of chr redundant genes in chromate resistance by LB400. It was found that B. xenovorans plant rhizosphere strains lacking the megaplasmid are chromate-sensitive, suggesting that the chr gene present in this replicon is responsible for the chromate-resistance phenotype of the LB400 strain. Transformation of a chromate-sensitive B. xenovorans strain with each of the six cloned LB400 chr genes showed that genes from 'adaptive replicons' (chrA1b and chr1NCb from chromosome 2 and chrA2 from the megaplasmid) conferred higher chromate resistance levels than chr genes from 'central' chromosome 1 (chrA1a, chrA6, and chr1NCa). An LB400 insertion mutant affected in the chrA2 gene displayed a chromate-sensitive phenotype, which was fully reverted by transferring the chrA2 wild-type gene, and partially reverted by chrA1b or chr1NCb genes. These data indicate that chr genes from adaptive replicons, mainly chrA2 from the megaplasmid, are responsible for the B. xenovorans LB400 chromate-resistance phenotype.

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA − mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA − mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4–5.3 times in the lexA − mutant, relative to transcription of the corresponding genes in the lexA + strain, but decreased significantly in the lexA − /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5′ regions of SOS genes, suggesting an indirect mechanism of regulation.

The chromate ion transporter (CHR) superfamily comprises transporters that confer chromate resistance by extruding toxic chromate ions from cytoplasm.Burkholderia xenovoransstrain LB400 has been reported to encode six CHR homologues in its multireplicon genome. We found that strain LB400 displays chromate-inducible resistance to chromate. Susceptibility tests ofEscherichia colistrains transformed with clonedB. xenovorans chrgenes indicated that the six genes confer chromate resistance, although under different growth conditions, and suggested that expression ofchrgenes is regulated by sulfate. Expression ofchrgenes was measured by quantitative reverse transcription-PCR (RT-qPCR) from total RNA ofB. xenovoransLB400 grown under different concentrations of sulfate and exposed or not to chromate. Thechrhomologues displayed distinct expression levels, but showed no significant differences in transcription under the various sulfate concentrations tested, indicating that sulfate does not regulatechrgene expression inB. xenovorans. ThechrA2gene, encoded in the megaplasmid, was the onlychrgene whose expression was induced by chromate and it was shown to constitute the chromate-responsivechrBACFoperon. These data suggest that this determinant is mainly responsible for theB. xenovoransLB400 chromate resistance phenotype.

The Bacillus subtilis strain 168 genome contains the chr3N-chr3C genes encoding the Chr3N/Chr3C protein pair of the chromate ion transporter (CHR) superfamily. Chr3N/Chr3C confers chromate resistance in Escherichia coli only when both proteins are expressed. Upstream of chr3N is the chrS gene encoding ChrS, a protein with homology to the Lrp/AsnC family of transcriptional regulators. When the chrS-chr3N-chr3C gene cluster was transferred to E. coli, a diminished level of chromate resistance was observed, as compared with E. coli transformants bearing only the chromate resistance genes, which displayed full resistance. These data suggested that the chrS gene product acts as negative regulator. RT-PCR assays demonstrated that expression of chrS diminishes transcription of the chromate resistance genes in E. coli, and that this repression was overcome by chromate. Electrophoretic mobility shift assays showed that purified ChrS protein specifically binds to the 5′ region of chrS. These results indicate that the chr gene cluster forms an operon regulated negatively by ChrS binding to its own gene’s regulatory region, and positively by chromate ions. Sequence analysis revealed similar operons in many Bacillales strains, suggesting some adaptive advantage. This is the first example of a bacterial heavy-metal resistance system controlled by an Lrp-type transcriptional regulator.

Short-chain monodomain family comprises pairs of membrane proteins of about 200 amino acid residues each that belong to the chromate ion transporter (CHR) superfamily. The short-chain CHR homologous pair Chr3N/Chr3C from Bacillus subtilis strain 168 confers chromate resistance only when both proteins are expressed. Membrane topology of the Chr3N and Chr3C proteins was determined in Escherichia coli by the analysis of translational fusions with reporter enzymes alkaline phosphatase and β-galactosidase. Each short-chain CHR protein was found to consist of five transmembrane segments with antiparallel orientation between them. The C terminus of Chr3N is located in the cytoplasm, whereas the C terminus of Chr3C is located in the periplasm. In silico analyses suggest that this antiparallel arrangement is shared by all protein members of the short-chain CHR3 subfamily and that the two Chr3N/Chr3C proteins might carry out distinct functions for the transport of chromate.

The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in México. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (Cr(R)), whereas 36% were resistant to mercury (Hg(R)). Cr(R) levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of Cr(R) isolates), encoding a CHR transporter homologue, and merA gene (74% of Hg(R) isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of Cr(R) isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred Cr(R), as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that Cr(R) genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.

The cyanobacterium Synechococcus elongatus strain PCC 7942 possesses pANL, a plasmid rich in genes related to sulfur metabolism. One of these genes, srpC, encodes the SrpC protein, a homologue of the CHR chromate ion transporter superfamily. The srpC gene was cloned and expressed in Escherichia coli and its role in relation to sulfate and chromate was analyzed. srpC was unable to complement the growth of an E. coli cysA sulfate uptake mutant when sulfate was utilized as a sole sulfur source, suggesting that SrpC is not a sulfate transporter. Expression of srpC in E. coli conferred chromate resistance and caused diminished chromate uptake. These results suggest that the S. elongatus SrpC protein functions as a transporter that extrudes chromate ions from the cell’s cytoplasm, and further demonstrate the close relationship between sulfate and chromate metabolism in this organism.

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.

The genomes of pseudomonad`species display a wide variety of adaptive genes which allow them to tolerate heavy metal toxicity. Several resistance systems have been directly characterized by genetic and biochemical tests, whereas other may only be inferred by comparison with the systems encoded in the genomes of other bacteria. This review briefly summarizes the mechanisms of resistance to heavy metals in pseudomonads. For this purpose, metals have been divided into three groups: (i) micronutrient cations that show toxicity (copper, cobalt, nickel, zinc); (ii) nonessential toxic cations (cadmium, lead, mercury, siliver); and (iii) toxic oxyanions (derived from arsenic, chromium, selenium and tellurium). Pseudomonads have evolved two main strategies to cope with heavy metal toxicity: membrane transporters able to efflux toxic ions from the cytoplasm, which include members of the major membrane protein families, and enzymatic redox detoxification pathways, which transform metals to less-toxic forms. Most heavy metal resistance systems in pseudomonads are encoded by complex operons, located on chromosomes or on plasmids, commonly involving delicate regulatory switches functioning at the transcriptional level. The abundance and diversity of genetic determinants conferring metal tolerance in the genomes of pseudomonads may be associated with the varied environments that these versatile bacteria inhabit.

Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily ( ywrB and ywrA ) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C , respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli , suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.