Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus

Resistance to the aminoglycosides gentamicin (Gmr), tobramycin (Tmr) and kanamycin (Kmr) in strains of Staphylococcus aureus isolated in Australia is mediated by the transposon Tn4001. The 1.35 kb inverted repeat of this transposon exhibits many of the characteristics of an insertion sequence and has consequently been designated IS256. Tandem duplication of IS256 contiguous with Tn4001 results in an increase in the level of GmrTmrKmr, thereby implying that the element possesses strong promoter sequences. Both contiguous and independent insertions of IS256 into the staphylococcal chromosome have been observed, the latter suggesting that the element may play a role in molecular rearrangements of the genome.     

IS15, a new insertion sequence widely spread in R plasmids of gram-negative bacteria

We have shown that the IS15 element, first detected in Salmonella ordonez and previously designated IS1522 (Labigne-Roussel et al. 1981), could transpose, with an approximate frequency of 5 X 10(-5), to various sites of different replicons in an Escherichia coli host deficient for general homologous recombination. Physical mapping with restriction endonucleases of this 1,500 base pairs (bp) transposable module indicated the presence of two, possibly contiguous, directly repeated internal sequences, at least 480 bp in size. IS15 could generate in vivo, by intramolecular recombination between the two direct repeats, IS15-delta, which is 830 bp in size. The reverse transition, IS15-delta to IS15, was not observed. The two related structural forms of IS15 were detected, by Southern hybridization, on plasmids belonging to various incompatibility groups (Inc6-C, I1, 7-M, and Y) isolated from phylogenetically remote pathogenic bacterial genera (Escherichia coli, Salmonella panama, Enterobacter cloacae, and Acinetobacter calcoaceticus). Whereas IS15 could promote its own transposition and transposition of DNA fragments it flanked, IS15-delta resulting from the 670 bp 'clean' deletion and representing the most common natural deletion derivative could only induce replicon fusion. It appears, therefore, that the two structural configurations of IS15 have evolved to play, by transposition, distinct and complementary roles in bacterial evolution.     

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.     

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.     

The electrophoretic mobility of gram-negative and gram-positive bacteria: an electrokinetic analysis.

The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser. Under standard growth conditions and neutral pH all cells displayed a negative EPM. The polysaccharide capsules of Escherichia coli strains K1, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM. Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67. No difference in the EPM was observed between rapidly growing and stationary-phase E. coli B. De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E. coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media. Extrusion of filamentous bacteriophage f1 from cells of its host, E. coli A95, caused a shift to a higher negative EPM. We also measured a variety of Gram-positive strains, all of which displayed different EPMs. When membrane fractions of E. coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed. The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.     

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.     

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements

Abstract The nucleotide sequence of IS 1126 , the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis , has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transpsosase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposashad homology to putative transposases of IS 1106 and IS 1186 both of which belong to the IS 5 group within the IS₄ super-family of insertion elements. On the basis of this homology we propose that IS 1126 should also be included in the IS 5 group. Southern-blot analysis of a number of P. gingivalis strains using IS 1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS 1126 from other closely related Porphyromonas species. This should allow IS 1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis .     

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.     

Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria.

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Escherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylococcus aureus were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain D-glutamate-adding activity and have Km and Vmax values similar to those of the regenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 microM for E. coli and H. influenzae MurD, respectively. Such substrate inhibition was not observed with the E. faecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO4(2-) had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.     

Distribution of insertion sequence ISRm1 in Rhizobium meliloti and other gram-negative bacteria

An internal 0.9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.     

Rapid methods for differentiating gram-positive from gram-negative aerobic and facultative anaerobic bacteria

Different tests based on lysis by KOH and on reaction with fluorogenic and chromogenic substrates, L-alanine-4-nitroanilide (LANA); L-alanine-4-methoxy- beta-naphthylamide (MNA); 4-alanine-2-amidoacridone (AAA); L-alanine-7-amido- 4-methylcoumarin (AAMC); 8-anilino-1-naphthalene-sulphonic acid (ANS) were compared for their suitability to distinguish Gram-positive from Gram-negative bacteria. A concentration of 100 micrograms/ml was chosen for incorporating LANA, AAA, AAMC and ANS into the growth medium, based on sensitivity tests. MNA did not show any detectable reaction over a concentration range from 50 to 200 micrograms/ml, and led to inhibition of all bacteria at 200 micrograms/ml. In the examination of a total of 146 bacterial strains, including Yersinia enterocolitica, Bacillus cereus, and B. subtilis the KOH test was not comparable with the Gram staining. A good correlation with Gram staining was found between LANA, AAA and AAMC added to plate count agar on one hand, and LANA and AAMC impregnated paper strips on the other hand, thereby utilizing the aminopeptidase activity. Agar containing ANS showed detectable fluorescence with all Gram-negative strains, but with Staphylococcus aureus and Staph. epidermidis a weak reaction was also observed. AAMC was selected for a rapid paper strip test. With this substrate a pronounced blue fluorescence was obtained with Gram-negative colonies.     

Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.     

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.     

Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans.

A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.     

IS801, an insertion sequence element isolated from Pseudomonas syringae pathovar phaseolicola

A transposable element, designated IS801, was isolated from strain LR781 of Pseudomonas syringae pathovar phaseolicola in two independent events using the entrapment plasmid, pUCD800. IS801 is 1517 base pairs in length and contains open reading frames that potentially encode proteins of 311 and 172 amino acids, as well as smaller proteins. Unlike most other prokaryotic transposable elements, IS801 lacks terminal repeats. Sequence analysis revealed two target pentamers for IS801 insertion that differ by one base pair. One copy of IS801 generated a perfect duplication of its target, TGAAC. The second copy of IS801 was flanked by the target, TGGAC, at one end, and TGAAC at the other end. A third copy of IS801 was cloned from pMMC7105, an indigenous plasmid of strain LR781, and it was flanked by copies of the pentamer TGAAC.     

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.     

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.