Maximal Sum of Metabolic Exchange Fluxes Outperforms Biomass Yield as a Predictor of Growth Rate of Microorganisms

Growth rate has long been considered one of the most valuable phenotypes that can be measured in cells. Aside from being highly accessible and informative in laboratory cultures, maximal growth rate is often a prime determinant of cellular fitness, and predicting phenotypes that underlie fitness is key to both understanding and manipulating life. Despite this, current methods for predicting microbial fitness typically focus on yields [e.g., predictions of biomass yield using GEnome-scale metabolic Models (GEMs)] or notably require many empirical kinetic constants or substrate uptake rates, which render these methods ineffective in cases where fitness derives most directly from growth rate. Here we present a new method for predicting cellular growth rate, termed SUMEX, which does not require any empirical variables apart from a metabolic network (i.e., a GEM) and the growth medium. SUMEX is calculated by maximizing the SUM of molar EXchange fluxes (hence SUMEX) in a genome-scale metabolic model. SUMEX successfully predicts relative microbial growth rates across species, environments, and genetic conditions, outperforming traditional cellular objectives (most notably, the convention assuming biomass maximization). The success of SUMEX suggests that the ability of a cell to catabolize substrates and produce a strong proton gradient enables fast cell growth. Easily applicable heuristics for predicting growth rate, such as what we demonstrate with SUMEX, may contribute to numerous medical and biotechnological goals, ranging from the engineering of faster-growing industrial strains, modeling of mixed ecological communities, and the inhibition of cancer growth.     

CTLA-4 Modulates the Differentiation of Inducible Foxp3+ Treg Cells but IL-10 Mediates Their Function in Experimental Autoimmune Encephalomyelitis

In vitro induced Foxp3⁺ T regulatory (iTreg) cells form a novel and promising target for therapeutic tolerance induction. However, the potential of these cells as a target for the treatment of various immune diseases, as well as the factors involved in their development and function, remain debated. Here, we demonstrate in a myelin basic protein (MBP)-specific murine model of CNS autoimmune disease that adoptive transfer of antigen-specific iTreg cells ameliorates disease progression. Moreover, we show that the co-stimulatory molecule CTLA-4 mediates in vitro differentiation of iTreg cells. Finally, we demonstrate that the secreted, immunosuppressive cytokine IL-10 controls the ability of antigen-specific iTreg cells to suppress autoimmune disease. Overall, we conclude that antigen-specific iTreg cells, which depend on various immune regulatory molecules for their differentiation and function, represent a major target for effective immunotherapy of autoimmune disease.     

Diverse lineages of pathogenic Leptospira species are widespread in the environment in Puerto Rico,USA

BackgroundLeptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology.Methodology/Principal findingsWe collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro.We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup.Conclusions/SignificanceDiverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.     

Genome of Enterobacteriophage Lula/phi80 and Insights into Its Ability To Spread in the Laboratory Environment

The novel temperate bacteriophage Lula, contaminating laboratory Escherichia coli strains, turned out to be the well-known lambdoid phage phi80. Our previous studies revealed that two characteristics of Lula/phi80 facilitate its spread in the laboratory environment: cryptic lysogen productivity and stealthy infectivity. To understand the genetics/genomics behind these traits, we sequenced and annotated the Lula/phi80 genome, encountering an E. coli-toxic gene revealed as a gap in the sequencing contig and analyzing a few genes in more detail. Lula/phi80''s genome layout copies that of lambda, yet homology with other lambdoid phages is mostly limited to the capsid genes. Lula/phi80''s DNA is resistant to cutting with several restriction enzymes, suggesting DNA modification, but deletion of the phage''s damL gene, coding for DNA adenine methylase, did not make DNA cuttable. The damL mutation of Lula/phi80 also did not change the phage titer in lysogen cultures, whereas the host dam mutation did increase it almost 100-fold. Since the high phage titer in cultures of Lula/phi80 lysogens is apparently in response to endogenous DNA damage, we deleted the only Lula/phi80 SOS-controlled gene, dinL. We found that dinL mutant lysogens release fewer phage in response to endogenous DNA damage but are unchanged in their response to external DNA damage. The toxic gene of Lula/phi80, gamL, encodes an inhibitor of the host ATP-dependent exonucleases, RecBCD and SbcCD. Its own antidote, agt, apparently encoding a modifier protein, was found nearby. Interestingly, Lula/phi80 lysogens are recD and sbcCD phenocopies, so GamL and Agt are part of lysogenic conversion.     

Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA

Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H₂O₂. Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death.     

All grown‐up and nowhere to go: paedomorphosis and local adaptation in Ambystoma salamanders in the Cuenca Oriental of Mexico

Facultative or obligate paedomorphosis has evolved several times in Mexican populations of the salamander genus Ambystoma, leading to increased genetic divergence among populations with alternate life histories and contributing to population divergence in this species complex. In the present study, we surveyed the genetic diversity of Ambystoma populations in lakes of the Cuenca Oriental, a high elevation closed drainage basin that encompasses permanent crater lakes harbouring salamander populations. We genotyped individuals from five populations aiming to better understand population dynamics and the evolution of paedomorphosis in this system. Specifically, we tested the hypotheses that the evolution of paedomorphosis in Ambystoma taylori resulted in reduced genetic exchange with populations of Ambystoma velasci in neighbouring lakes. Second, we tested whether the populations in brackish lakes of the Cuenca Oriental, Lake Atexcac, and Lake Alchichica show restricted gene flow across the basin, possibly as a result of local adaptation to those microhabitats. Using various indices of population genetic diversity, Bayesian assignment, and approximate Bayesian computation methods, we show that genetic exchange between brackish lakes and freshwater lakes is negligible, despite continued gene flow among freshwater lakes. We show that the first divergence among populations occurred between Alchichica and the remaining populations and that the evolution of paedomorphosis in A. taylori was likely favoured by local adaptation to saline conditions, thus increasing its genetic isolation. An apparently similar process appears to be in progress independently in lake Atexcac, showing that local adaptation may play an important role in population isolation and, ultimately, in speciation.     

Epidemiological Surveillance of Serratia marcescens Infections by Bacteriocin Typing

During 10 months, 155 isolates of Serratia marcescens were cultured from 105 patients, of whom 49 were considered to have significant infection. The 155 isolates were typed by bacteriocin sensitivity, and 137 (88.4%) were assigned to 37 provisional bacteriocin groups; 18 isolates were nontypable. No major outbreaks of nosocomial infection were demonstrable; however, there were four chronologically separate minor episodes of cross-infection that involved two or three patients per room or unit, respectively.