PIMMS (Pragmatic Insertional Mutation Mapping System) laboratory methodology a readily accessible tool for identification of essential genes in Streptococcus

The Pragmatic Insertional Mutation Mapping (PIMMS) laboratory protocol was developed alongside various bioinformatics packages (Blanchard et al., 2015) to enable detection of essential and conditionally essential genes in Streptococcus and related bacteria. This extended the methodology commonly used to locate insertional mutations in individual mutants to the analysis of mutations in populations of bacteria. In Streptococcus uberis, a pyogenic Streptococcus associated with intramammary infection and mastitis in ruminants, the mutagen pGhost9:ISS1 was shown to integrate across the entire genome. Analysis of >80,000 mutations revealed 196 coding sequences, which were not be mutated and a further 67 where mutation only occurred beyond the 90th percentile of the coding sequence. These sequences showed good concordance with sequences within the database of essential genes and typically matched sequences known to be associated with basic cellular functions. Due to the broad utility of this mutagen and the simplicity of the methodology it is anticipated that PIMMS will be of value to a wide range of laboratories in functional genomic analysis of a wide range of Gram positive bacteria (Streptococcus, Enterococcus, and Lactococcus) of medical, veterinary, and industrial significance

Genes Important for Catalase Activity in Enterococcus faecalis

Little in general is known about how heme proteins are assembled from their constituents in cells. The Gram-positive bacterium Enterococcus faecalis cannot synthesize heme and does not depend on it for growth. However, when supplied with heme in the growth medium the cells can synthesize two heme proteins; catalase (KatA) and cytochrome bd (CydAB). To identify novel factors important for catalase biogenesis libraries of E. faecalis gene insertion mutants were generated using two different types of transposons. The libraries of mutants were screened for clones deficient in catalase activity using a colony zymogram staining procedure. Analysis of obtained clones identified, in addition to katA (encoding the catalase enzyme protein), nine genes distributed over five different chromosomal loci. No factors with a dedicated essential role in catalase biogenesis or heme trafficking were revealed, but the results indicate the RNA degradosome (srmB, rnjA), an ABC-type oligopeptide transporter (oppBC), a two-component signal transducer (etaR), and NADH peroxidase (npr) as being important for expression of catalase activity in E. faecalis. It is demonstrated that catalase biogenesis in E. faecalis is independent of the CydABCD proteins and that a conserved proline residue in the N-terminal region of KatA is important for catalase assembly

Heme Protein Biogenesis - Catalase in Enterococcus faecalis

Heme proteins form a large and diverse group of proteins which are involved in a variety of biological functions. The heme prosthetic group enables them to carry out redox reactions, transport electrons, bind gaseous molecules, and function as sensors. Despite their importance only little is generally known about heme protein biogenesis. The Gram-positive bacterium Enterococcus faecalis is found in the gastrointestinal tract of mammals and is an opportunistic pathogen. E. faecalis cannot synthesize heme and does not require heme for growth. When supplied with heme, this bacterium produces two heme proteins; one catalase and one cytochrome bd. These properties of E. faecalis have been exploited in this work to study the physiological role and biogenesis of catalase. Catalase was found to contribute to resistance against exogenous and endogenous hydrogen peroxide stress. It is shown that the gene for catalase, katA, is expressed independently of heme in the growth medium. KatA protein was found in cells growing in heme-free medium but was degraded in stationary growth phase unless heme was supplied. These and other findings were used to devise a procedure for the purification of apo-catalase polypeptide. It is demonstrated in vitro with isolated apo-catalase and heme that catalase can be de novo assembled. The obtained catalase contained stoichiometric amounts of heme but did not show full enzyme activity. These and other results suggested that the in vitro assembled catalase is stalled at an intermediate state and that one or more soluble cell factors are needed to complete assembly or activation of the enzyme. To find novel factors important for catalase assembly, two constructed libraries of transposon-insertion mutants were screened for catalase deficient mutants. In this way ten independent katA mutations were isolated but no factors (genes) with a dedicated essential role for catalase biogenesis or heme trafficking were revealed. However, the screen indicated nine genes, distributed over five different chromosomal loci, which apparently indirectly affect expression of catalase in E. faecalis

Heme proteins in lactic Acid bacteria.

Lactic acid bacteria (LAB) are of profound importance in food production and infection medicine. LAB do not rely on heme (protoheme IX) for growth and are unable to synthesize this cofactor but are generally able to assemble a small repertoire of heme-containing proteins if heme is provided from an exogenous source. These features are in contrast to other bacteria, which synthesize their heme or depend on heme for growth. We here present the cellular function of heme proteins so far identified in LAB and discuss their biogenesis as well as applications of the extraordinary heme physiology of LAB

Production, purification and detergent exchange of isotopically labeled Bacillus subtilis cytochrome b(558) (SdhC)

Cytochrome 6558 of the gram-positive bacterium Bacillus subtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinella succinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b(558). In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome 6558 produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. (C) 2011 Elsevier Inc. All rights reserved

Contribution of catalase to hydrogen peroxide resistance in Enterococcus faecalis.

Enterococcus faecalis exhibits high resistance to oxidative stress. Several enzymes are responsible for this trait. The role of alkyl hydroperoxide reductase (Ahp), thiol peroxidase (Tpx), and NADH peroxidase (Npr) in oxidative stress defense was recently characterized. Enterococcus faecalis, in contrast to many other streptococci, contains a catalase (KatA), but this enzyme can only be formed when the bacterium is supplied with heme. We have used this heme dependency of catalase activity and mutants deficient in KatA and Npr to investigate the role of the catalase in resistance against exogenous and endogenous hydrogen peroxide stress. The results demonstrate that in the presence of environmental heme catalase contributes to the protection against toxic effects of hydrogen peroxide

In Vitro Assembly of Catalase.

Most aerobic organisms contain catalase which functions to decompose hydrogen peroxide. Typical catalases are structurally complex homo-tetrameric enzymes with one heme prosthetic group buried in each subunit. It is not known how catalase in the cell is assembled from its constituents. The bacterium Enterococcus faecalis cannot synthesize heme but can acquire it from the environment to form a cytoplasmic catalase. We have in E. faecalis monitored production of the enzyme polypeptide (KatA) depending on the availability of heme and used our findings to devise a procedure for the purification of preparative amounts of in vivo-synthesized apocatalase. We show that fully active catalase can be obtained in vitro by incubating isolated apoprotein with hemin. We have characterized features of the assembly process and describe a temperature-trapped hemylated intermediate of the enzyme maturation process. Hemylation of apocatalase does not require auxiliary cell components but rapid assembly of active enzyme seemingly is assisted in the cell. Our findings provide insight about catalase assembly and offer new experimental possibilities for detailed studies of this process

Loci for which two or more independent catalase-deficient transposon insertion mutants were obtained.

<p>In the screen for catalase deficiency two or more independent transposon insertions were obtained in six loci. Catalase activity and KatA content in cell extracts are expressed as fraction relative to those of the parental strain OG1RF.</p><p><i>nd</i>, not done; <i>na</i>, not applicable.</p>a<p>GenBank: CP002621.1.</p>b<p>Catalase activity of strain OG1RF was 14 U/mg of protein.</p>c<p>IS<i>S1</i> system.</p

Bacterial strains.

<p>Bacterial strains.</p