The CYP134 family in Gram-positive bacteria: From Bacillus to beyond, an orphan P450 lineage awaiting functional discovery.

The mRNA level of the aconitase gene acn of Corynebacterium glutamicum is reduced under iron limitation. Here we show that an AraC-type regulator, termed RipA for "regulator of iron proteins A," is involved in this type of regulation. A C. glutamicum DeltaripA mutant has a 2-fold higher aconitase activity than the wild type under iron limitation, but not under iron excess. Comparison of the mRNA profiles of the DeltaripA mutant and the wild type revealed that the acn mRNA level was increased in the DeltaripA mutant under iron limitation, but not under iron excess, indicating a repressor function of RipA. Besides acn, some other genes showed increased mRNA levels in the DeltaripA mutant under iron starvation (i.e. those encoding succinate dehydrogenase (sdhCAB), nitrate/nitrite transporter and nitrate reductase (narKGHJI), isopropylmalate dehydratase (leuCD), catechol 1,2-dioxygenase (catA), and phosphotransacetylase (pta)). Most of these proteins contain iron. Purified RipA binds to the upstream regions of all operons mentioned above and in addition to that of the catalase gene (katA). From 13 identified binding sites, the RipA consensus binding motif RRGCGN(4)RYGAC was deduced. Expression of ripA itself is repressed under iron excess by DtxR, since purified DtxR binds to a well conserved binding site upstream of ripA. Thus, repression of acn and the other target genes indicated above under iron limitation involves a regulatory cascade of two repressors, DtxR and its target RipA. The modulation of the intracellular iron usage by RipA supplements mechanisms for iron acquisition that are directly regulated by DtxR.

Engineering cytochrome P450 enzyme systems for biomedical and biotechnological applications

Is Mycobacterium tuberculosis a closer relative to Gram-positive or Gram–negative bacterial pathogens?

Integrative conjugative elements (ICEs) occur frequently in Gram-positive and Gram-negative bacteria. In contrast to plasmids, they are stably integrated in the bacterial genome, often inserted in a tRNA gene. They are excised from the host chromosome upon induction in order to be transferred to a recipient cell. When conjugative transfer is completed, they stably reintegrate in the chromosome. It is generally thought that ICEs are incapable of autonomous replication, instead relying on replication and segregation along with the host chromosome. In this issue of Molecular Microbiology Lee and co-workers demonstrate that ICEBs1 from Bacillus subtilis is capable of autonomous plasmid-like replication in its circular form after excision. The authors show that ICEBs1 replication is unidirectional; it initiates at oriT(ICEBs1) and requires the ICEBs1-encoded conjugative relaxase NicK. Replication also requires the catalytic subunit of the host DNA polymerase PolC, the host processivity clamp DnaN and the host-encoded alternative helicase PcrA. Autonomous replication of ICEBs1 appears to be important for its stable maintenance, but not for horizontal transfer of the element. Lee and co-workers argue that plasmid-like replication is likely a common property of ICEs, probably contributing to stability and maintenance of ICEs in bacterial populations. I discuss these findings in context with data on other ICEs from Gram-positive and Gram-negative bacteria and with respect to possible consequences of the findings for basic research on mobile genetic elements from Gram-positive bacteria and their applications in biotechnology.

ABSTRACTBurkholderia pseudomallei is an opportunistic pathogen that is responsible for the disease melioidosis in humans and animals. The microbe is a tier 1 select agent because it is highly infectious by the aerosol route, it is inherently resistant to multiple antibiotics, and no licensed vaccine currently exists. Naturally acquired infections result from contact with contaminated soil or water sources in regions of endemicity. There have been few reports investigating the molecular mechanism(s) utilized by B. pseudomallei to survive and persist in ecological niches harboring microbial competitors. Here, we report the isolation of Gram-positive bacteria from multiple environmental sources and show that ∼45% of these isolates are inhibited by B. pseudomallei in head-to-head competition assays. Two competition-deficient B. pseudomallei transposon mutants were identified that contained insertion mutations in the hmqA-G operon. This large biosynthetic gene cluster encodes the enzymes that produce a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). Liquid chromatography and mass spectrometry conducted on filter-sterilized culture supernatants revealed five HMAQs and N-oxide derivatives that were produced by the parental strain but were absent in an isogenic hmqD deletion mutant. The results demonstrate that B. pseudomallei inhibits the growth of environmental Gram-positive bacteria in a contact-independent manner via the production of HMAQs by the hmqA-G operon.IMPORTANCEBurkholderia pseudomallei naturally resides in water, soil, and the rhizosphere and its success as an opportunistic pathogen is dependent on the ability to persist in these harsh habitats long enough to come into contact with a susceptible host. In addition to adapting to limiting nutrients and diverse chemical and physical challenges, B. pseudomallei also has to interact with a variety of microbial competitors. Our research shows that one of the ways in which B. pseudomallei competes with Gram-positive environmental bacteria is by exporting a diverse array of closely related antimicrobial secondary metabolites.

Recently, resistance of pathogens towards conventional antibiotics has increased, representing a threat to public health globally. As part of the fight against this, studies on alternative antibiotics such as antimicrobial peptides have been performed, and it has been shown that their sequence and structure are closely related to their antimicrobial activity. Against this background, we here evaluated the antibacterial activity of two peptides developed by solid-phase synthesis, Alyteserin 1c (WT) and its mutant derivative (ΔM), which shows increased net charge and reduced hydrophobicity. These structural characteristics were modified as a result of amino acid substitutions on the polar face of the WT helix. The minimum inhibitory concentration (MIC) of both peptides was obtained in Gram-positive and Gram-negative bacteria. The results showed that the rational substitutions of the amino acids increased the activity in Gram-positive bacteria, especially against Staphylococcus aureus, for which the MIC was one-third of that for the WT analog. In contrast to the case for Gram-positive bacteria, these substitutions decreased activity against Gram-negative bacteria, especially in Escherichia coli, for which the MIC was eight-fold higher than that exhibited by the WT peptide. To understand this, models of the peptide behavior upon interacting with membranes of E. coli and S. aureus created using molecular dynamics were studied and it was determined that the helical stability of the peptide is indispensable for antimicrobial activity. The hydrogen bonds between the His20 of the peptides and the phospholipids of the membranes should modulate the selectivity associated with structural stability at the carboxy-terminal region of the peptides.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.

<p>Net primary production is a major contributor to carbon export in the Southern Ocean and supports rich marine ecosystems [Henley et al., 2020], driven in part by high macronutrient availability and summertime light levels, but ultimately constrained by seasonal changes in light and scarce supply of the essential micronutrient iron [Martin et al., 1990; Boyd, 2002; Tagliabue et al., 2016]. Although changing iron stress is a component of climate-driven trends in model projections of net primary production [Bopp et al., 2013; Laufkotter et al., 2015; Kwiatkowski et al., 2020], our confidence in the accuracy of their predictions is undermined by a lack of <em>in situ</em> constraints at appropriate spatial and temporal scales [Tagliabue et al., 2016; Tagliabue et al., 2020]. Earth System Models tend to predict increased Southern Ocean net primary production by the end of the 21st century, but are characterized by significant inter-model disagreement [Bopp et al., 2013; Kwiatkowski et al., 2020 Biogeosciences].  We show a significant multi-decadal increase in <em>in situ</em> iron stress from 1996 to 2020 that is positively correlated to the Southern Annular Mode and reflected by diminishing <em>in situ</em> net primary production over the last five years. It is not possible to directly infer Fe stress from observed concentrations, which necessitate experimental approaches (<em>in situ</em> open ocean fertilization / bottle nutrient addition experiments or proteomics). These experimental methods cannot be easily applied at appropriate spatial and temporal scales across the Southern Ocean that are required to assess trends in ecosystem status linked to climate drivers. Our novel proxy for <em>in situ</em> iron stress is based on the degree of non-photochemical quenching in relation to available light as a measurable photophysiological response to iron availability [Alderkamp et al., 2019; Schuback & Tortell, 2019; Schallenberg et al., 2020; Ryan-Keogh & Thomalla, 2020]. The proxy was able to reproduce expected variations in iron stress that occur seasonally [Boyd, 2002] and from natural and artificial fertilization [Boyd et al., 2000; Coale et al., 2004; Blain et al., 2008]. A particular strength of this iron stress proxy is that it can be retrospectively applied to data from ships and autonomous platforms with coincident measurements of fluorescence, photosynthetically active radiation and backscatter or beam attenuation to deliver a long-term time series. An iron stress trend of this magnitude in the Southern Ocean, where the primary constraint on net primary production is known to be iron limitation, is likely to have significant implications for the effectiveness of the biological carbon pump globally and may impact the trajectory of climate. The progressive <em>in situ</em> trend of increasing iron stress is however much stronger than net primary production trends from a suite of remote sensing and earth system models, indicating hitherto potential underestimation of ongoing Southern Ocean change.</p>

Past: The title ‘PTS 50 or The PTS after 50 years' relies on the first description in 1964 of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system (PTS) by Kundig, Gosh and Roseman [Proc Natl Acad Sci USA 1964;52:1067-1074]. The system comprised proteins named Enzyme I, HPr and Enzymes II, as part of a novel PTS for carbohydrates in Gram-negative and Gram-positive bacteria, whose ‘biological significance remained unclear'. In contrast, studies which would eventually lead to the discovery of the central role of the PTS in bacterial metabolism had been published since before 1942. They are primarily linked to names like Epps and Gale, J. Monod, Cohn and Horibata, and B. Magasanik, and to phenomena like ‘glucose effects', ‘diauxie', ‘catabolite repression' and carbohydrate transport. Present: The pioneering work from Roseman's group initiated a flood of publications. The extraordinary progress from 1964 to this day in the qualitative and in vitro description of the genes and enzymes of the PTS, and of its multiple roles in global cellular control through ‘inducer exclusion', gene induction and ‘catabolite repression', in cellular growth, in cell differentiation and in chemotaxis, as well as the differences of its functions between Gram-positive and Gram-negative bacteria, was one theme of the meeting and will not be treated in detail here. Future: At the 1988 Paris meeting entitled ‘The PTS after 25 years', Saul Roseman predicted that ‘we must describe these interactions [of the PTS components] in a quantitative way [under] in vivo conditions'. I will present some results obtained by our group during recent years on the old phenomenon of diauxie by means of very fast and quantitative tests, measured in vivo, and obtained from cultures of isogenic mutant strains growing under chemostat conditions. The results begin to hint at the problems relating to future PTS research, but also to the ‘true science' of Roseman.

Rhamnosyltransferases: Biochemical activities, potential biotechnology for production of natural products and their applications.

BackgroundThe metabolic processing of ellagic acid (EA) by cytochrome P450s (CYP450s) expressed in the intestines is unclear. This study aimed to investigate the effects of CYP450s that are highly expressed in HIEC cells on metabolic activity of EA.Material/MethodsHIEC cell models expressing 2B6, 2C9, 2D6, and 3A4 were generated by stably transfecting with CYP450 genes using a lentivirus system. PCR and Western blot assay were used to detect expression of CYP450s. Cell Counting Kit-8 (CCK-8) assay was used to examine the cytotoxic effect of EA on CYP450s-expressing HIEC cells. Flow cytometry was employed to evaluate apoptosis of CYP450s-expressing HIEC cells after addition of EA. Metabolic clearance rate of EA in vitro by the constructed HIEC cell models was measured using UPLC-MS method.ResultsCYP450s expression HIEC cell models, including CYP2B6, CYP2C9, CYP2D6, and CYP3A4, were successfully established. EA treatment at different concentrations (10 μg/mL and 50 μg/mL) remarkably decreased cell viability of HIEC cells expressing CYP2C9 compared to the untreated control (p<0.01), in a concentration-dependent and time-dependent manner. Expression of CYP2C9 significantly increased the apoptosis rate of HIEC cells treated with EA compared to that in HIEC cells without any CYP450s expression (p<0.01). The clearance rate of EA in CYP2B6-expressing (p<0.05) and CYP2C9-expressing (p<0.001) HIEC cell models was remarkably reduced after 120 min.ConclusionsEllagic acid was effectively activated by CYP2C9 in HIEC cells and caused cytotoxicity and apoptosis of HIEC cells. Therefore, CYP2C9 is main metabolic enzyme of EA when compared to other CYP450 HIEC cell models.

Steroid compounds are produced by eukaryotes where they have a variety of chemical structures and play important physiological roles. Many bacteria are capable of transforming and completely degrading steroids under various growth conditions. The microbial metabolism of steroids has gained considerable interest due to its potential applications in industrial and environmental biotechnology. The oxic degradation pathways of steroids and some of the involved enzymes are well characterized. The key players in these pathways are oxygenases which depend on dioxygen as a co-substrate. On the contrary, much less is known about the mechanisms operating under anoxic conditions. Obviously, anoxic bacterial metabolism of steroids should proceed via oxygenase-independent reactions. So far, a few bacteria that can completely degrade steroids in the absence of oxygen were characterized. Surprisingly, all of them belong to denitrifying bacteria and utilize only nitrate as the alternative electron acceptor. Recent studies of anoxic metabolism of steroids using denitrifying bacteria revealed unique and interesting biochemical reactions and enzymes. Here we discuss the current understanding of the biochemistry and molecular biology of bacterial steroid metabolism under anoxic conditions. The aerobic metabolism of steroids is briefly presented for the sake of comparison. Future investigations on anoxic metabolism of steroids will unravel novel aspects of the regulation and evolution of catabolic pathways as well as unprecedented biocatalysts with useful applications in biotechnology.

Editor's evaluation: Resource allocation accounts for the large variability of rate-yield phenotypes across bacterial strains

Biophysical, photochemical and biochemical characterization of a protease from Aspergillus tamarii URM4634

Iron limitation is one major constraint of microbial life, and a plethora of microbes use siderophores for high affinity iron acquisition. Because specific enzymes for reductive iron release in gram-positives are not known, we searched Firmicute genomes and found a novel association pattern of putative ferric siderophore reductases and uptake genes. The reductase from the schizokinen-producing alkaliphile Bacillus halodurans was found to cluster with a ferric citrate-hydroxamate uptake system and to catalyze iron release efficiently from Fe[III]-dicitrate, Fe[III]-schizokinen, Fe[III]-aerobactin, and ferrichrome. The gene was hence named fchR for ferric citrate and hydroxamate reductase. The tightly bound [2Fe-2S] cofactor of FchR was identified by UV-visible, EPR, CD spectroscopy, and mass spectrometry. Iron release kinetics were determined with several substrates by using ferredoxin as electron donor. Catalytic efficiencies were strongly enhanced in the presence of an iron-sulfur scaffold protein scavenging the released ferrous iron. Competitive inhibition of FchR was observed with Ga(III)-charged siderophores with K(i) values in the micromolar range. The principal catalytic mechanism was found to couple increasing K(m) and K(D) values of substrate binding with increasing k(cat) values, resulting in high catalytic efficiencies over a wide redox range. Physiologically, a chromosomal fchR deletion led to strongly impaired growth during iron limitation even in the presence of ferric siderophores. Inductively coupled plasma-MS analysis of ΔfchR revealed intracellular iron accumulation, indicating that the ferric substrates were not efficiently metabolized. We further show that FchR can be efficiently inhibited by redox-inert siderophore mimics in vivo, suggesting that substrate-specific ferric siderophore reductases may present future targets for microbial pathogen control.