Presence of growth factors for Methanobacterium ruminantium in both gram-positive and gram-negative bacteria

Autoclaved cells of gram-positive bacteria or mixed rumen organisms promote the growth of rumen strains of Methanobacterium ruminantium, but cells of E. coli were only stimulatory to growth after treatment with lysozyme plus EDTA or with EDTA alone.N-acetylglucosamine is identified as one of the growth factors for rumen strains of Mb. ruminantium.     

Electrochemical classification of gram-negative and gram-positive bacteria

Intestinal bacteria were classified as gram-positive or gram-negative by an electrode system with a basal plane pyrolytic graphite electrode and a porous nitrocellulose membrane filter to trap bacteria. When the potential of the graphite electrode was run in the range of 0 to 1.0 V versus the saturated calomel electrode (SCE), gram-positive bacteria gave peak currents at 0.65 to 0.69 V versus the SCE. The peak potentials of gram-negative bacteria were 0.70 to 0.74 V versus the SCE. Gram-negative bacteria and gram-positive bacteria were also classified based on the ratio of the second peak current to the first peak current when the potential cycle was repeated twice. The numbers of cells on the membrane filter were determined from the peak currents. It was found that the peak currents result from the electrochemical oxidation of coenzyme A in the cells of Escherichia coli and Lactobacillus acidophilus.     

Evidence that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen.

It was demonstrated that mycoplasmas, gram-negative bacteria, and certain gram-positive bacteria share a similar protein antigen with a molecular weight ranging from 42,000 to 48,000. Western blotting (immunoblotting) with an antibody specific to a 43-kDa membrane protein of Mycoplasma fermentans showed the existence of this protein antigen in all Mycoplasma spp. tested (14 species), Acholeplasma laidlawii (1 strain), and gram-negative bacteria (8 species) but only in Staphylococcus aureus of four gram-positive species tested. Neither Ureaplasma urealyticum nor mammalian cell cultures showed any cross-reactions with this antibody. These proteins were found in both cytoplasmic and membrane fractions of mycoplasma cells but were not exposed on the surface of mycoplasmal or bacterial cells.     

A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.     

High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria.

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.     

Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Cadmium uptake by growing cells of gram-positive and gram-negative bacteria

The present study evaluates the effect of the cadmium (Cd2+) on the growth and protein synthesis of some Gram-positive (Staphylococcus aureus, Bacillus subtilis and Streptococcus faecium) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and the cadmium uptake by the same micro-organisms. The Gram-negative bacteria tested were less sensitive to metal ions than the Gram-positive, and P. aeruginosa was the most resistant. The Gram-negative bacteria were also able to accumulate higher amounts of cadmium during growth than the Gram-positive bacteria. The maximum values of specific metal uptake (microgram of Cd2+ incorporated per mg of protein) were: 0.52 for S. aureus, 0.65 for S. faecium, 0.79 for B. subtilis, 2.79 for E. coli and 24.15 for P. aeruginosa, respectively. The differences in the ability to accumulate metal found between Gram-negative and Gram-positive bacteria seems to account for different mechanisms of metal resistance.     

Rapid method for distinction of gram-negative from gram-positive bacteria

Summary A rapid method for distinction between gram-negative and grampositive bacteria by means of a 3% solution of potassium hydroxide is tested on 71 gram-positive and 55 gram-negative bacterial strains. The method proved reliable with one exception only, a Bacillus macerans strain. That strain was definately gram-negative on staining. Other Bacillus strains were proved gram-positive by the test, even those being gram-negative on staining.     

Functional analysis of alkane hydroxylases from gram-negative and gram-positive bacteria

We have cloned homologs of the Pseudomonas putida GPo1 alkane hydroxylase from Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens CHA0, Alcanivorax borkumensis AP1, Mycobacterium tuberculosis H37Rv, and Prauserella rugosa NRRL B-2295. Sequence comparisons show that the level of protein sequence identity between the homologs is as low as 35%, and that the Pseudomonas alkane hydroxylases are as distantly related to each other as to the remaining alkane hydroxylases. Based on the observation that rubredoxin, an electron transfer component of the GPo1 alkane hydroxylase system, can be replaced by rubredoxins from other alkane hydroxylase systems, we have developed three recombinant host strains for the functional analysis of the novel alkane hydroxylase genes. Two hosts, Escherichia coli GEc137 and P. putida GPo12, were equipped with pGEc47 Delta B, which encodes all proteins necessary for growth on medium-chain-length alkanes (C(6) to C(12)), except a functional alkane hydroxylase. The third host was an alkB knockout derivative of P. fluorescens CHA0, which is no longer able to grow on C(12) to C(16) alkanes. All alkane hydroxylase homologs, except the Acinetobacter sp. ADP1 AlkM, allowed at least one of the three hosts to grow on n-alkanes.     

Electron microscopic features of gram-negative and gram-positive bacteria embedded in phosphotungstate

Gram-negative bacteria belonging to different families show a rugose surface structure, which is absent in gram-positive bacteria. Mesosomes and cytoplasmic inclusions with normal and anomalous contrast are demonstrated in gram-positive bacteria.     

Genetic regulation of plasmid transfer in gram-negative bacteria

The data are reviewed on the genetic structure and regulation of tra-genes activity controlling the conjugative transfer of F, F-like plasmids as well as the transfer of some other bacterial plasmids. The effect of the systems inhibiting the conjugational transfer (fin-systems) of F and a number of derepressed F-like plasmids has been characterized on the basis of data obtained by the authors or published data. Possible mechanisms for the systems functioning in the regulation of tra-genes activity are discussed as well as the prospects of their further study.     

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.     

Protamine-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria.

The inhibitory effect of the cationic peptide protamine on Listeria monocytogenes, Escherichia coli, and Shewanella putrefaciens has been studied in detail. The addition of protamine (10 to 1,000 micrograms/ml) resulted in inhibition of oxygen consumption after less than 1 min and loss of intracellular carboxyfluorescein and ATP after 2 to 5 min. Maximum antibacterial activity was reached at alkaline pH and in the absence of divalent cations. The efficient permeabilization of cell envelopes of both gram-positive and gram-negative bacteria suggests that protamine causes a general disruption of the cell envelope, leading to a rapid and nonspecific efflux of low- and high-molecular-weight compounds.     

Metabolism of o-phthalic acid by different gram-negative and gram-positive soil bacteria

Different bacteria, isolated from soil by the enrichment method, were able to grow on phthalic acid as carbon source. Protocatechuate was identified as intermediate in phthalate metabolism. All phthalategrown bacteria oxidized phthalate and protocatechuate rapidly without having a lag-period. Benzoic acid, terephthalic acid, protocatechuic acid, salicylic acid, di- and mono-butyl phthalate were also metabolized by some of the organisms, benzoic acid being degraded via catechol and terephthalic acid via protocatechuate as intermediate. All organisms tested cleaved protocatechuate or catechol, respectively, by the ortho fission, when grown on phthalate, terephthalate, or benzoate as carbon source. A characterization and tentative identification of the organisms is given.     

Human peptidoglycan recognition proteins require zinc to kill both gram-positive and gram-negative bacteria and are synergistic with antibacterial peptides

Mammals have four peptidoglycan recognition proteins (PGRPs or PGLYRPs), which are secreted innate immunity pattern recognition molecules with effector functions. In this study, we demonstrate that human PGLYRP-1, PGLYRP-3, PGLYRP-4, and PGLYRP-3:4 have Zn(2+)-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria at physiologic Zn(2+) concentrations found in serum, sweat, saliva, and other body fluids. The requirement for Zn(2+) can only be partially replaced by Ca(2+) for killing of Gram-positive bacteria but not for killing of Gram-negative bacteria. The bactericidal activity of PGLYRPs is salt insensitive and requires N-glycosylation of PGLYRPs. The LD(99) of PGLYRPs for Gram-positive and Gram-negative bacteria is 0.3-1.7 muM, and killing of bacteria by PGLYRPs, in contrast to killing by antibacterial peptides, does not involve permeabilization of cytoplasmic membrane. PGLYRPs and antibacterial peptides (phospholipase A(2), alpha- and beta-defensins, and bactericidal permeability-increasing protein), at subbactericidal concentrations, synergistically kill Gram-positive and Gram-negative bacteria. These results demonstrate that PGLYRPs are a novel class of recognition and effector molecules with broad Zn(2+)-dependent bactericidal activity against both Gram-positive and Gram-negative bacteria that are synergistic with antibacterial peptides.     

Comparative surface-to-hand and fingertip-to-mouth transfer efficiency of gram-positive bacteria,gram-negative bacteria,and phage

AIMS: To determine the transfer efficiency of micro-organisms from fomites to hands and the subsequent transfer from the fingertip to the lip. METHODS AND RESULTS: Volunteers hands were sampled after the normal usage of fomites seeded with a pooled culture of a Gram-positive bacterium (Micrococcus luteus), a Gram-negative bacterium (Serratia rubidea) and phage PRD-1 (Period A). Activities included wringing out a dishcloth/sponge, turning on/off a kitchen faucet, cutting up a carrot, making hamburger patties, holding a phone receiver, and removing laundry from the washing machine. Transfer efficiencies were 38.47% to 65.80% and 27.59% to 40.03% for the phone receiver and faucet, respectively. Transfer efficiencies from porous fomites were <0.01%. In most cases, M.luteus was transferred most efficiently, followed by phage PRD-1 and S. rubidea. When the volunteers' fingertips were inoculated with the pooled organisms and held to the lip area (Period B), transfer rates of 40.99%, 33.97%, and 33.90% occurred with M. luteus, S. rubidea, and PRD-1, respectively. CONCLUSIONS: The highest bacteral transfer rates from fomites to the hands were seen with the hard, non-porous surfaces. Even with low transfer rates, the numbers of bacteria transferred to the hands were still high (up to 10(6) cells). Transfer of bacteria from the fingertip to the lip is similar to that observed from hard surfaces to hands. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious doses of pathogens may be transferred to the mouth after handling an everyday contaminated household object.