- Volume 164, Issue 4, 2018

The yeast Pichia pastoris (syn. Komagataella spp.) is a popular cell factory for recombinant protein production. Yeasts in general provide a good starting point for cell factory engineering. They are intrinsically robust and easy to manipulate and cultivate. However, their secretory pathway is not evolutionarily adapted to high loads of secretory protein. In particular, more complex proteins, like the antibody fragment (Fab) used in this study, overwhelm the folding and secretion capacity. This triggers cellular stress responses, which may cause excessive intracellular degradation. Previous results have shown that, in fact, about 60 % of the newly synthesized Fab is intracellularly degraded. Endoplasmic reticulum-associated protein degradation (ERAD) is one possible intracellular degradation pathway for proteins aimed for secretion. We therefore targeted ERAD for cell factory engineering and investigated the impact on recombinant protein secretion in P. pastoris. Three components of the ERAD-L complex, which is involved in the degradation of luminal proteins, and a protein involved in proteasomal degradation, were successfully disrupted in Fab-secreting P. pastoris. Contrary to expectation, the effect on secretion was marginal. In the course of more detailed investigation of the impact of ERAD, we took a closer look at the intracellular variants of the recombinant protein. This enabled us to further zero in on the issue of intracellular Fab degradation and exclude an overshooting ER quality control. We propose that a major fraction of the Fab is actually degraded before entering the secretory pathway.

Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of ‘anti-CRISPR’ proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the ‘gene drive’. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.

Shiga toxin-producing Escherichia coli (STEC) are characterized by the release of potent Shiga toxins (Stx), which are associated with severe intestinal and renal disease. Although all STEC strains produce Stx, only a few serotypes cause infection in humans. To determine which virulence traits in vitro are linked to human disease in vivo, 13 Stx2a-producing STEC strains of seropathotype (SPT) A or B (associated with severe human intestinal disease and outbreaks) and 6 strains of SPT D or E (rarely or not linked to human disease) were evaluated in a microaerobic human colonic epithelial infection model. All SPT strains demonstrated similar growth, colonization of polarized T84 colon carcinoma cells and Stx release into the medium. In contrast, Stx translocation across the T84 cell monolayer was significantly lower in SPT group DE compared to SPT group AB strains. Further experiments showed that Stx penetration occurred via a transcellular pathway and was independent of bacterial type III secretion and attaching and effacing lesion formation. These results suggest that the extent of Stx transcytosis across the gut epithelium may represent an important indicator of STEC pathogenicity for humans.

The hyd1/hyd2 hydrophobins are important constituents of the conidial cell wall of the insect pathogenic fungus Beauveria bassiana. This fungus can also form intimate associations with several plant species. Here, we show that inactivation of two Class I hydrophobin genes, hyd1 or hyd2, significantly decreases the interaction of B. bassiana with bean roots. Curiously, the ∆hyd1/∆hyd2 double mutant was less impaired in root association than Δhyd1 or Δhyd2. Loss of hyd genes affected growth rate, conidiation ability and oosporein production. Expression patterns for genes involved in conidiation, cell wall integrity, insect virulence, signal transduction, adhesion, hydrophobicity and oosporein production were screened in the deletion mutants grown in different conditions. Repression of the major MAP-Kinase signal transduction pathways (Slt2 MAPK pathway) was observed that was more pronounced in the single versus double hyd mutants under certain conditions. The ∆hyd1/∆hyd2 double mutant showed up-regulation of the Hog1 MAPK and the Msn2 transcription factor under certain conditions when compared to the wild-type or single hyd mutants. The expression of the bad2 adhesin and the oosporein polyketide synthase 9 gene was severely reduced in all of the mutants. On the other hand, fewer changes were observed in the expression of key conidiation and cell wall integrity genes in hyd mutants compared to wild-type. Taken together, the data from this study indicated pleiotropic consequences of deletion of hyd1 and hyd2 on signalling and stress pathways as well as the ability of the fungus to form stable associations with plant roots.

Porphyromonas gingivalis produces hydrogen sulfide (H2S) from l-cysteine. However, the role of H2S produced by P. gingivalis in periodontal inflammation is unclear. In this study, we identified the enzyme that catalyses H2S production from l-cysteine and analysed the role of H2S using a mouse abscess model. The enzyme identified was identical to methionine γ-lyase (PG0343), which produces methyl mercaptan (CH3SH) from l-methionine. Therefore, we analysed H2S and CH3SH production by P. gingivalis W83 and a PG0343-deletion mutant (ΔPG0343) with/without l-cysteine and/or l-methionine. The results indicated that CH3SH is produced constitutively irrespective of the presence of l-methionine, while H2S was greatly increased by both P. gingivalis W83 and ΔPG0343 in the presence of l-cysteine. In contrast, CH3SH production by ΔPG0343 was absent irrespective of the presence of l-methionine, and H2S production was eliminated in the absence of l-cysteine. Thus, CH3SH and H2S production involves different substrates, l-methionine or l-cysteine, respectively. Based on these characteristics, we analysed the roles of CH3SH and H2S in abscess formation in mice by P. gingivalis W83 and ΔPG0343. Abscess formation by P. gingivalis W83, but not ΔPG0343, differed significantly in the presence and absence of l-cysteine. In addition, the presence of l-methionine did not affect the size of abscesses generated by P. gingivalis W83 and ΔPG0343. Therefore, we conclude that H2S produced by P. gingivalis does not induce inflammation; however, H2S enhances inflammation caused by CH3SH. Thus, these results suggest the H2S produced by P. gingivalis plays a supportive role in inflammation caused by methionine γ-lyase.

Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.

Synthesis of the Escherichia coli YtfE protein, also known as RIC, for the repair of damaged iron centres, is highly induced during anaerobic growth under conditions of nitrosative stress. How YtfE repairs nitrosative damage remains unclear. Contrary to previous reports, we show that strains defective in YtfE that lack the high-affinity NO reductase activity of the hybrid cluster protein (Hcp) are less sensitive to nitrosative stress than isogenic ytfE+ strains, which are extremely sensitive. Evidence that this sensitivity is due to YtfE-dependent release of NO into the cytoplasm includes: relief of growth inhibition by PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), which degrades NO; relief of nitrosative stress by deletion of narG encoding the nitrate reductase that is the major source of NO from nitrite; partial suppression of nitrosative stress due to loss of Hcp function by a further mutation in ytfE; YtfE-dependent loss of aconitase and fumarase activities in the absence of Hcp; and YtfE-dependent relief of NsrR repression of the hcp promoter in response to cytoplasmic NO. We suggest that a major role for YtfE is to reverse nitrosative damage by releasing, directly or indirectly, NO from nitrosylated proteins into the cytoplasm where the high-affinity NO reductase activity of Hcp ensures its reduction to N2O. If so, the concerted action of YtfE and Hcp would not only maintain the cytoplasmic concentration of NO in the low nM range, but also provide a rationalization for the coordinate regulation of Hcp and YtfE synthesis by NsrR.

Carbonic anhydrase (CA) EcaA of Synechococcus elongatus PCC 7942 was previously characterized as a putative extracellular α-class CA, however, its activity was never verified. Here we show that EcaA possesses specific CA activity, which is inhibited by ethoxyzolamide. An active EcaA was expressed in heterologous bacterial system, which supports the formation of disulfide bonds, as a full-length protein (EcaA+L) and as a mature protein that lacks a leader peptide (EcaA-L). EcaA-L exhibited higher specific activity compared to EcaA+L. The recombinant EcaA, expressed in a bacterial system that does not support optimal disulfide bond formation, exhibited extremely low activity. This activity, however, could be enhanced by the thiol-oxidizing agent, diamide; while a disulfide bond-reducing agent, dithiothreitol, further inactivated the enzyme. Intact E. coli cells that overexpress EcaA+L possess a small amount of processed protein, EcaA-L, whereas the bulk of the full-length protein resides in the cytosol. This may indicate poor recognition of the EcaA leader peptide by protein export systems. S. elongatus possessed a relatively low level of ecaA mRNA, which varied insignificantly in response to changes in CO2 supply. However, the presence of protein in the cells is not obvious. This points to the physiological insignificance of EcaA in S. elongatus, at least under the applied experimental conditions.

The aceA and glcB genes, encoding isocitrate lyase (ICL) and malate synthase, respectively, are not in an operon in many bacteria, including Pseudomonas aeruginosa, unlike in Escherichia coli. Here, we show that expression of aceA in P. aeruginosa is specifically upregulated under H2O2-induced oxidative stress and under iron-limiting conditions. In contrast, the addition of exogenous redox active compounds or antibiotics increases the expression of glcB. The transcriptional start sites of aceA under iron-limiting conditions and in the presence of iron were found to be identical by 5′ RACE. Interestingly, the enzymatic activities of ICL and isocitrate dehydrogenase had opposite responses under different iron conditions, suggesting that the glyoxylate shunt (GS) might be important under iron-limiting conditions. Remarkably, the intracellular iron concentration was lower while the iron demand was higher in the GS-activated cells growing on acetate compared to cells growing on glucose. Absence of GS dysregulated iron homeostasis led to changes in the cellular iron pool, with higher intracellular chelatable iron levels. In addition, GS mutants were found to have higher cytochrome c oxidase activity on iron-supplemented agar plates of minimal media, which promoted the growth of the GS mutants. However, deletion of the GS genes resulted in higher sensitivity to a high concentration of H2O2, presumably due to iron-mediated killing. In conclusion, the GS system appears to be tightly linked to iron homeostasis in the promotion of P. aeruginosa survival under oxidative stress.

Polyamines (PAs) are ubiquitous polycations derived from basic l-amino acids whose physiological roles are still being defined. Their biosynthesis and functions in nitrogen-fixing rhizobia such as Sinorhizobium meliloti have not been extensively investigated. Thin layer chromatographic and mass spectrometric analyses showed that S. meliloti Rm8530 produces the PAs, putrescine (Put), spermidine (Spd) and homospermidine (HSpd), in their free forms and norspermidine (NSpd) in a form bound to macromolecules. The S. meliloti genome encodes two putative ornithine decarboxylases (ODC) for Put synthesis. Activity assays with the purified enzymes showed that ODC2 (SMc02983) decarboxylates both ornithine and lysine. ODC1 (SMa0680) decarboxylates only ornithine. An odc1 mutant was similar to the wild-type in ODC activity, PA production and growth. In comparison to the wild-type, an odc2 mutant had 45 % as much ODC activity and its growth rates were reduced by 42, 14 and 44 % under non-stress, salt stress or acid stress conditions, respectively. The odc2 mutant produced only trace levels of Put, Spd and HSpd. Wild-type phenotypes were restored when the mutant was grown in cultures supplemented with 1 mM Put or Spd or when the odc2 gene was introduced in trans. odc2 gene expression was increased under acid stress and reduced under salt stress and with exogenous Put or Spd. An odc1 odc2 double mutant had phenotypes similar to the odc2 mutant. These results indicate that ODC2 is the major enzyme for Put synthesis in S. meliloti and that PAs are required for normal growth in vitro.

Actinomycete bacteria use polyprenol phosphate mannose as a lipid linked sugar donor for extra-cytoplasmic glycosyl transferases that transfer mannose to cell envelope polymers, including glycoproteins and glycolipids. We showed recently that strains of Streptomyces coelicolor with mutations in the gene ppm1 encoding polyprenol phosphate mannose synthase were both resistant to phage φC31 and have greatly increased susceptibility to antibiotics that mostly act on cell wall biogenesis. Here we show that mutations in the genes encoding enzymes that act upstream of Ppm1 in the polyprenol phosphate mannose synthesis pathway can also confer phage resistance and antibiotic hyper-susceptibility. GDP-mannose is a substrate for Ppm1 and is synthesised by GDP-mannose pyrophosphorylase (GMP; ManC) which uses GTP and mannose-1-phosphate as substrates. Phosphomannomutase (PMM; ManB) converts mannose-6-phosphate to mannose-1-phosphate. S. coelicolor strains with knocked down GMP activity or with a mutation in sco3028 encoding PMM acquire phenotypes that resemble those of the ppm1 - mutants i.e. φC31 resistant and susceptible to antibiotics. Differences in the phenotypes of the strains were observed, however. While the ppm1 - strains have a small colony phenotype, the sco3028 :: Tn5062 mutants had an extremely small colony phenotype indicative of an even greater growth defect. Moreover we were unable to generate a strain in which GMP activity encoded by sco3039 and sco4238 is completely knocked out, indicating that GMP is also an important enzyme for growth. Possibly GDP-mannose is at a metabolic branch point that supplies alternative nucleotide sugar donors.

Polyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for Rhodosprillum rubrum, raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type R. rubrum with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules’ associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. In silico analysis revealed that Q2RSI4 harbours a Phasin_2 family domain and shares low identity with a single-strand DNA-binding protein from Sphaerochaeta coccoides. Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.

The opportunistic pathogen Pseudomonas aeruginosa can metabolize carnitine and O-acylcarnitines, which are abundant in host muscle and other tissues. Acylcarnitines are metabolized to carnitine and a fatty acid. The liberated carnitine and its catabolic product, glycine betaine, can be used as osmoprotectants, to induce the secreted phospholipase C PlcH, and as sole carbon, nitrogen and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines, therefore they must be imported from an exogenous source. In this study, we present the first characterization of bacterial acylcarnitine transport. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines (MCACs and LCACs) are hydrolysed extracytoplasmically and the free carnitine is transported primarily through CaiX-CbcWV. These findings suggest that the periplasmic protein CaiX has a binding pocket that permits short acyl chains on its carnitine ligand and that there are one or more secreted hydrolases that cleave MCACs and LCACs. To identify the secreted hydrolase(s), we used a saturating genetic screen and transcriptomics followed by phenotypic analyses, but neither led to identification of a contributing hydrolase, supporting but not conclusively demonstrating redundancy for this activity.