Vṛtti and Vṛttikāra in Rāmānuja’s Śrī-bhāṣya

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Interference with histidyl-tRNA synthetase by a CRISPR spacer sequence as a factor in the evolution of Pelobacter carbinolicus

BACKGROUND: Pelobacter carbinolicus, a bacterium of the family Geobacteraceae, cannot reduce Fe(III) directly or produce electricity like its relatives. How P. carbinolicus evolved is an intriguing problem. The genome of P. carbinolicus contains clustered regularly interspaced short palindromic repeats (CRISPR) separated by unique spacer sequences, which recent studies have shown to produce RNA molecules that interfere with genes containing identical sequences. RESULTS: CRISPR spacer #1, which matches a sequence within hisS, the histidyl-tRNA synthetase gene of P. carbinolicus, was shown to be expressed. Phylogenetic analysis and genetics demonstrated that a gene paralogous to hisS in the genomes of Geobacteraceae is unlikely to compensate for interference with hisS. Spacer #1 inhibited growth of a transgenic strain of Geobacter sulfurreducens in which the native hisS was replaced with that of P. carbinolicus. The prediction that interference with hisS would result in an attenuated histidyl-tRNA pool insufficient for translation of proteins with multiple closely spaced histidines, predisposing them to mutation and elimination during evolution, was investigated by comparative genomics of P. carbinolicus and related species. Several ancestral genes with high histidine demand have been lost or modified in the P. carbinolicus lineage, providing an explanation for its physiological differences from other Geobacteraceae. CONCLUSIONS: The disappearance of multiheme c-type cytochromes and other genes typical of a metal-respiring ancestor from the P. carbinolicus lineage may be the consequence of spacer #1 interfering with hisS, a condition that can be reproduced in a heterologous host. This is the first successful co-introduction of an active CRISPR spacer and its target in the same cell, the first application of a chimeric CRISPR construct consisting of a spacer from one species in the context of repeats of another species, and the first report of a potential impact of CRISPR on genome-scale evolution by interference with an essential gene

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

BACKGROUND: The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences. RESULTS: The experimentally observed greater metabolic versatility of G. metallireducens versus G. sulfurreducens is borne out by the presence of more numerous genes for metabolism of organic acids including acetate, propionate, and pyruvate. Although G. metallireducens lacks a dicarboxylic acid transporter, it has acquired a second putative succinate dehydrogenase/fumarate reductase complex, suggesting that respiration of fumarate was important until recently in its evolutionary history. Vestiges of the molybdate (ModE) regulon of G. sulfurreducens can be detected in G. metallireducens, which has lost the global regulatory protein ModE but retained some putative ModE-binding sites and multiplied certain genes of molybdenum cofactor biosynthesis. Several enzymes of amino acid metabolism are of different origin in the two species, but significant patterns of gene organization are conserved. Whereas most Geobacteraceae are predicted to obtain biosynthetic reducing equivalents from electron transfer pathways via a ferredoxin oxidoreductase, G. metallireducens can derive them from the oxidative pentose phosphate pathway. In addition to the evidence of greater metabolic versatility, the G. metallireducens genome is also remarkable for the abundance of multicopy nucleotide sequences found in intergenic regions and even within genes. CONCLUSION: The genomic evidence suggests that metabolism, physiology and regulation of gene expression in G. metallireducens may be dramatically different from other Geobacteraceae

Construction of a Geobacter Strain With Exceptional Growth on Cathodes

Insoluble extracellular electron donors are important sources of energy for anaerobic respiration in biogeochemical cycling and in diverse practical applications. The previous lack of a genetically tractable model microorganism that could be grown to high densities under anaerobic conditions in pure culture with an insoluble extracellular electron donor has stymied efforts to better understand this form of respiration. We report here on the design of a strain of Geobacter sulfurreducens, designated strain ACL, which grows as thick (ca. 35 μm) confluent biofilms on graphite cathodes poised at -500 mV (versus Ag/AgCl) with fumarate as the electron acceptor. Sustained maximum current consumption rates were >0.8 A/m2, which is >10-fold higher than the current consumption of the wild-type strain. The improved function on the cathode was achieved by introducing genes for an ATP-dependent citrate lyase, completing the complement of enzymes needed for a reverse TCA cycle for the synthesis of biosynthetic precursors from carbon dioxide. Strain ACL provides an important model organism for elucidating the mechanisms for effective anaerobic growth with an insoluble extracellular electron donor and may offer unique possibilities as a chassis for the introduction of synthetic metabolic pathways for the production of commodities with electrons derived from electrodes

Deltaproteobacteria (Pelobacter) and Methanococcoides are responsible for choline-dependent methanogenesis in a coastal saltmarsh sediment

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment

Novel regulatory cascades controlling expression of nitrogen-fixation genes in Geobacter sulfurreducens

Geobacter species often play an important role in bioremediation of environments contaminated with metals or organics and show promise for harvesting electricity from waste organic matter in microbial fuel cells. The ability of Geobacter species to fix atmospheric nitrogen is an important metabolic feature for these applications. We identified novel regulatory cascades controlling nitrogen-fixation gene expression in Geobacter sulfurreducens. Unlike the regulatory mechanisms known in other nitrogen-fixing microorganisms, nitrogen-fixation gene regulation in G. sulfurreducens is controlled by two two-component His–Asp phosphorelay systems. One of these systems appears to be the master regulatory system that activates transcription of the majority of nitrogen-fixation genes and represses a gene encoding glutamate dehydrogenase during nitrogen fixation. The other system whose expression is directly activated by the master regulatory system appears to control by antitermination the expression of a subset of the nitrogen-fixation genes whose transcription is activated by the master regulatory system and whose promoter contains transcription termination signals. This study provides a new paradigm for nitrogen-fixation gene regulation

CRISPR Inhibition of Prophage Acquisition in Streptococcus pyogenes

Streptococcus pyogenes, one of the major human pathogens, is a unique species since it has acquired diverse strain-specific virulence properties mainly through the acquisition of streptococcal prophages. In addition, S. pyogenes possesses clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems that can restrict horizontal gene transfer (HGT) including phage insertion. Therefore, it was of interest to examine the relationship between CRISPR and acquisition of prophages in S. pyogenes. Although two distinct CRISPR loci were found in S. pyogenes, some strains lacked CRISPR and these strains possess significantly more prophages than CRISPR harboring strains. We also found that the number of spacers of S. pyogenes CRISPR was less than for other streptococci. The demonstrated spacer contents, however, suggested that the CRISPR appear to limit phage insertions. In addition, we found a significant inverse correlation between the number of spacers and prophages in S. pyogenes. It was therefore suggested that S. pyogenes CRISPR have permitted phage insertion by lacking its own spacers. Interestingly, in two closely related S. pyogenes strains (SSI-1 and MGAS315), CRISPR activity appeared to be impaired following the insertion of phage genomes into the repeat sequences. Detailed analysis of this prophage insertion site suggested that MGAS315 is the ancestral strain of SSI-1. As a result of analysis of 35 additional streptococcal genomes, it was suggested that the influences of the CRISPR on the phage insertion vary among species even within the same genus. Our results suggested that limitations in CRISPR content could explain the characteristic acquisition of prophages and might contribute to strain-specific pathogenesis in S. pyogenes

Impact of Small Repeat Sequences on Bacterial Genome Evolution

Intergenic regions of prokaryotic genomes carry multiple copies of terminal inverted repeat (TIR) sequences, the nonautonomous miniature inverted-repeat transposable element (MITE). In addition, there are the repetitive extragenic palindromic (REP) sequences that fold into a small stem loop rich in G–C bonding. And the clustered regularly interspaced short palindromic repeats (CRISPRs) display similar small stem loops but are an integral part of a complex genetic element. Other classes of repeats such as the REP2 element do not have TIRs but show other signatures. With the current availability of a large number of whole-genome sequences, many new repeat elements have been discovered. These sequences display diverse properties. Some show an intimate linkage to integrons, and at least one encodes a small RNA. Many repeats are found fused with chromosomal open reading frames, and some are located within protein coding sequences. Small repeat units appear to work hand in hand with the transcriptional and/or post-transcriptional apparatus of the cell. Functionally, they are multifaceted, and this can range from the control of gene expression, the facilitation of host/pathogen interactions, or stimulation of the mammalian immune system. The CRISPR complex displays dramatic functions such as an acquired immune system that defends against invading viruses and plasmids. Evolutionarily, mobile repeat elements may have influenced a cycle of active versus inactive genes in ancestral organisms, and some repeats are concentrated in regions of the chromosome where there is significant genomic plasticity. Changes in the abundance of genomic repeats during the evolution of an organism may have resulted in a benefit to the cell or posed a disadvantage, and some present day species may reflect a purification process. The diverse structure, eclectic functions, and evolutionary aspects of repeat elements are described

Reduction of hydrogen peroxide in gram-negative bacteria – bacterial peroxidases

This work was supported by Fundação para a Ciência e Tecnologia (FCT) (project grants to SRP, PTDC/BIA-PRO/109796/2009 and PTDC/BIA-BQM/29442/2017, and a scholarship to CSN, SFRH/BD/87878/2012). Unidade de Ciências Biomoleculares Aplicadas-UCIBIO was financed by national funds from FCT/MEC (UID/Multi/04378/2019).Bacteria display an array of enzymes to detoxify reactive oxygen species that cause damage to DNA and to other biomolecules leading to cell death. Hydrogen peroxide is one of these species, with endogenous and exogenous sources, such as lactic acid bacteria, oxidative burst of the immune system or chemical reactions at oxic-anoxic interfaces. The enzymes that detoxify hydrogen peroxide will be the focus of this review, with special emphasis on bacterial peroxidases that reduce hydrogen peroxide to water. Bacterial peroxidases are periplasmic cytochromes with either two or three c-type haems, which have been classified as classical and non-classical bacterial peroxidases, respectively. Most of the studies have been focus on the classical bacterial peroxidases, showing the presence of a reductive activation in the presence of calcium ions. Mutagenesis studies have clarified the catalytic mechanism of this enzyme and were used to propose an intramolecular electron transfer pathway, with far less being known about the intermolecular electron transfer that occurs between reduced electron donors and the enzyme. The physiological function of these enzymes was not very clear until it was shown, for the non-classical bacterial peroxidase, that this enzyme is required for the bacteria to use hydrogen peroxide as terminal electron acceptor under anoxic conditions. These non-classical bacterial peroxidases are quinol peroxidases that do not require reductive activation but need calcium ions to attain maximum activity and share similar catalytic intermediates with the classical bacterial peroxidases.preprintpublishe