Phagocytosis influences the intracellular survival of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> via the endoplasmic reticulum stress response

Background

Mycobacterium smegmatis, a rapidly growing non-tuberculosis mycobacterium, is a good model for studying the pathogenesis of tuberculosis because of its genetic similarity to Mycobacterium tuberculosis (Mtb). Macrophages remove mycobacteria during an infection. Macrophage apoptosis is a host defense mechanism against intracellular bacteria. We have reported that endoplasmic reticulum (ER) stress is an important host defense mechanism against Mtb infection.

Results

In this study, we found that M. smegmatis induced strong ER stress. M. smegmatis-induced reactive oxygen species (ROS) play a critical role in the induction of ER stress-mediated apoptosis. Pretreatment with an ROS scavenger suppressed M. smegmatis-induced ER stress. Elimination of ROS decreased the ER stress response and significantly increased the intracellular survival of M. smegmatis. Interestingly, inhibition of phagocytosis significantly decreased ROS synthesis, ER stress response induction, and cytokine production.

Conclusions

Phagocytosis of M. smegmatis induces ROS production, leading to production of proinflammatory cytokines. Phagocytosis-induced ROS is associated with the M. smegmatis-mediated ER stress response in macrophages. Therefore, phagocytosis plays a critical role in the induction of ER stress-mediated apoptosis during mycobacterial infection.

     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Decreased formation of branched-chain short fatty acids in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> by metabolic engineering

Objectives

To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared.

Results

By knockout of the ptb gene, 1.01 g BCFAs kg^?1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg^?1). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg^?1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12.

Conclusions

HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

     

Metabolic engineering of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and in silico investigation for enhanced 2,3-butanediol production

Objectives

To improve the production of 2,3-butanediol (2,3-BD) in Klebsiella pneumoniae, the genes related to the formation of lactic acid, ethanol, and acetic acid were eliminated.

Results

Although the cell growth and 2,3-BD production rates of the K. pneumoniae ΔldhA ΔadhE Δpta-ackA strain were lower than those of the wild-type strain, the mutant produced a higher titer of 2,3-BD and a higher yield in batch fermentation: 91 g 2,3-BD/l with a yield of 0.45 g per g glucose and a productivity of 1.62 g/l.h in fed-batch fermentation. The metabolic characteristics of the mutants were consistent with the results of in silico simulation.

Conclusions

K. pneumoniae knockout mutants developed with an aid of in silico investigation could produce higher amounts of 2,3-BD with increased titer, yield, and productivity.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

Enhanced pyruvate production in <Emphasis Type="Italic">Candida glabrata</Emphasis> by overexpressing the <Emphasis Type="Italic">CgAMD1</Emphasis> gene to improve acid tolerance

Objectives

To enhance acid tolerance of Candida glabrata for pyruvate production by engineering AMP metabolism.

Results

The physiological function of AMP deaminase in AMP metabolism from C. glabrata was investigated by deleting or overexpresseing the corresponding gene, CgAMD1. At pH 4, CgAMD1 overexpression resulted in 59 and 51% increases in biomass and cell viability compared to those of wild type strain, respectively. In addition, the intracellular ATP level of strain Cgamd1Δ/CgAMD1 was down-regulated by 22%, which led to a 94% increase in pyruvate production. Further, various strengths of CgAMD1 expression cassettes were designed, thus resulting in a 59% increase in pyruvate production at pH 4. Strain Cgamd1Δ/CgAMD1 _(H) was grown in a 30 l batch bioreactor at pH 4, and pyruvate reached 46.1 g/l.

Conclusion

CgAMD1 overexpression plays an active role in improving acid tolerance and pyruvate fermentation performance of C. glabrata at pH 4.

     

Expression of lycopene biosynthesis genes fused in line with Shine-Dalgarno sequences improves the stress-tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

Lycopene biosynthetic genes from Deinococcus radiodurans were co-expressed in Lactococcus lactis to produce lycopene and improve its tolerance to stress.

Results

Lycopene-related genes from D. radiodurans, DR1395 (crtE), DR0862 (crtB), and DR0861 (crtI), were fused in line with S hine-Dalgarno (SD) sequences and co-expressed in L. lactis. The recombinant strain produced 0.36 mg lycopene g^-1 dry cell wt after 48 h fermentation. The survival rate to UV irradiation of the recombinant strain was higher than that of the non-transformed strain.

Conclusion

The L. lactis with co-expressed genes responsible for lycopene biosynthesis from D. radiodurans produced lycopene and exhibited increased resistance to UV stress, suggesting that the recombinant strain has important application potential in food industry.

     

Genome-wide analysis and stress-responsive expression of CCCH zinc finger family genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Background

Ubiquitous CCCH nucleic acid-binding motif is found in a wide-variety of organisms. CCCH genes are involved in plant developmental processes and biotic and abiotic stress responses. Brassica rapa is a vital economic crop and classical model plant of polyploidy evolution, but the functions of CCCH genes in B. rapa are unclear.

Results

In this study, 103 CCCH genes in B. rapa were identified. A comparative analysis of the chromosomal position, gene structure, domain organization and duplication event between B. rapa and Arabidopsis thaliana were performed. Results showed that CCCH genes could be divided into 18 subfamilies, and segmental duplication might mainly contribute to this family expansion. C-X_7/8-C-X₅-C₃-H was the most commonly found motif, but some novel CCCH motifs were also found, along with some loses of typical CCCH motifs widespread in other plant species. The multifarious gene structures and domain organizations implicated functional diversity of CCCH genes in B. rapa. Evidence also suggested functional redundancy in at least one subfamily due to high conservation between members. Finally, the expression profiles of subfamily-IX genes indicated that they are likely involved in various stress responses.

Conclusion

This study provides the first genome-wide characterization of the CCCH genes in B. rapa. The results suggest that B. rapa CCCH genes are likely functionally divergent, but mostly involved in plant development and stress response. These results are expected to facilitate future functional characterization of this potential RNA-binding protein family in Brassica crops.

     

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).

Results

Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.

Conclusions

Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

     

ClpXP affects the cell metabolism of <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> partially in an RpoS-dependent manner

Introduction

ClpXP protease is an important proteolytic system in Salmonella enterica serovar typhimurium (S. typhimurium). Inactivation of ClpXP by deletion of clpP resulted in overproduction of RpoS and a growth defect phenotype. Only one report has indicated that deleting rpoS can restore the growth of a S. typhimurium clpP mutant to the wild-type level. Whether overproduction of RpoS is responsible for the growth deficiency resulting from clpP disruption and how ClpXP affects the cell metabolism of S. typhimurium remain to be elucidated.

Objectives

The aim of this study is to investigate the effect of ClpXP on cell metabolism of S. typhimurium and explore the possible co-effect of RpoS associated with ClpXP in cell metabolism.

Method

We constructed a clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) using a two-step phage transduction technique. We then compared the metabolite fingerprints of Salmonella rpoS deletion mutant TT-14 (ΔrpoS TT-1), clpP deletion mutant TT-16 (ΔclpP TT-1), and clpP rpoS double deletion mutant TT-19 (ΔclpP ΔrpoS TT-1) with those of the wild-type strain TT-1 by using gas chromatography coupled with mass spectrometry (GC–MS).

Results

Deletion of rpoS recovered only a part of the growth of Salmonella clpP mutant. Further metabolome analysis indicated that clpP disruption changed the levels of 16 extra- and 19 intracellular substances, while the extracellular concentrations of 4 compounds (serine, l-5-oxoproline, l-glutamic acid, and l-tryptophan) and intracellular concentrations of 10 compounds (l-isoleucine, glycine, serine, l-methionine, l-phenylalanine, malic acid, citric acid, urea, putrescine, and 6-hydroxypurine) returned to their wild-type levels when rpoS was also deleted.

Conclusion

ClpXP affects the cell metabolism of S. typhimurium partially in an RpoS-dependent manner.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

Adherent/invasive <Emphasis Type="Italic">Escherichia coli</Emphasis> (AIEC) isolates from asymptomatic people: new <Emphasis Type="Italic">E. coli</Emphasis> ST131 O25:H4/H30-Rx virotypes

Background

The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried.

Methods

Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli CTX^R ST131 O25:H4/H30-Rx strains collected from healthy donors’ stool. The virulence genes patterns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated.

Findings

Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples.

Interpretation

The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors.

     

Hybrid incompatibilities in interspecific crosses between tetraploid wheat and its wild diploid relative <Emphasis Type="Italic">Aegilops umbellulata</Emphasis>

Key message

Herbaspirillum rubrisubalbicans decreases growth of rice. Inoculation of rice with H. rubrisubalbicansincreased the ACCO mRNA levels and ethylene production. The H. rubrisubalbicans riceinteractions were further characterized by proteomic approach.

Abstract

Herbaspirillum rubrisubalbicans is a well-known growth-promoting rhizobacteria that can also act as a mild phyto-pathogen. During colonisation of rice, RT-qPCR analyses showed that H. rubrisubalbicans up-regulates the methionine recycling pathway as well as phyto-siderophore synthesis genes. mRNA levels of ACC oxidase and ethylene levels also increased in rice roots but inoculation with H. rubrisubalbicans impaired growth of the rice plant. A proteomic approach was used to identify proteins specifically modulated by H. rubrisubalbicans in rice and amongst the differentially expressed proteins a V-ATPase and a 14-3-3 protein were down-regulated. Several proteins of H. rubrisubalbicans were identified, including the type VI secretion system effector Hcp1, suggesting that protein secretion play a role colonisation in rice. Finally, the alkyl hydroperoxide reductase, a primary scavenger of endogenous hydrogen peroxide was also identified. Monitoring the levels of reactive oxygen species in the epiphytic bacteria by flow cytometry revealed that H. rubrisubalbicans is subjected to oxidative stress, suggesting that the alkyl hydroperoxide reductase is an important regulator of redox homeostasis in plant-bacteria interactions.