Efficacy of silver treated catheters for haemodialysis in preventing bacterial adhesion.

The growing resistance of many strains of bacteria to antibiotics and antiseptics is becoming a serious problem in medicine. Nano-silver is one of the most prominent products in medicine because it exhibits unusual physicochemical properties and a strong biological activity. In this work an innovative silver deposition technology was applied to temporary polyurethane catheters for haemodialysis. The working conditions of catheters were reproduced through laboratory equipment that ensured the flow of deionized water and simulated body fluid inside the lumina at corporeal temperature. The growth and the adhesion of Staphylococcus aureus on the surface of the device were studied through fluorescence microscopy. ICP-AES was adopted to calculate the amount of silver released from the substrate. The stability of the coating during the whole working life of the device was demonstrated through thermo-gravimetric analysis.

Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons.

We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

Alternative patterns of his operon transcription and mRNA processing generated by metabolic perturbation.

Previous studies have shown that the expression of the his operon of Salmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA.

Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae.

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154-158 bp) and internally rearranged (104-108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26-27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.

Evolution and function of the neisserial dam-replacing gene.

Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis. In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5'-GmeATC-3'. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5'-GATC-3', similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.

The histidine operon of Azospirillum brasilense: organization, nucleotide sequence and functional analysis.

A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced. The sequence analysis revealed the presence of six major contiguous open reading frames (ORF). The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E. coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products. S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A. brasilense his operon. Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator. Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A. brasilense his cluster are discussed.

Scattering phenomena effects on growth of 308-nm laser-irradiated bacteria in suspension.

In this study we analyzed the effect of 308-nm laser exposure on recovery of irradiated Staphylococcus epidermidis held in liquid after irradiation and before plating. Coexistence of bacterial growth inhibition and stimulation phenomena was observed. Under certain conditions, bacterial recovery was about fivefold higher in irradiated samples than in the controls. The available evidence suggests that the growth inhibition was due to the bactericidal activity of the 308-nm wavelength light, whereas the growth stimulation effect was associated with broadband radiation generated by scattering phenomena in the bacterial suspensions. Spectroscopic investigations revealed that the nutrient broth plays a decisive role in the scattering of laser radiation within the suspension.

The respiratory chains of four strains of the alkaliphilic Bacillus clausii.

A comparative analysis of terminal respiratory enzymes has been performed on four strains of Bacillus clausii used for preparation of a European probiotic. These four strains originated most probably from a common ancestor through early selection of stable clones for industrial propagation. They exhibit a low level of intra-specific diversity and a high degree of genomic conservation, making them an attractive model to study the different bioenergetics behaviors of alkaliphilic bacilli. The analysis of the different bioenergetics responses has been carried out revealing striking differences among the strains. Two out of the four strains have shown a functional redundancy of the terminal part of the respiratory chain. The biochemical data correlate with the expression level of the mRNA of cytochrome c oxidase and quinol oxidase genes (heme-copper type). The consequences of these different bioenergetics behaviors are also discussed.

Epibiotic Vibrio luminous bacteria isolated from some hydrozoa and bryozoa species.

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA-DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.

Vibrio harveyi associated with Aglaophenia octodonta (Hydrozoa, Cnidaria).

A previously unknown association between a luminous bacterium, Vibrio harveyi, and a benthic hydrozoan, Aglaophenia octodonta, is described. Aglaophenia hydrocladia showed a clear fluorescence in the folds along the hydrocaulus and at the base of the hydrotheca, suggesting the presence of luminous bacteria. This hypothesis was confirmed by isolation of luminous bacteria from Aglaophenia homogenates. Phenotypic characterization of bacterial isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a single species: V. harveyi. The association between V. harveyi and A. octodonta has epidemiological as well as ecological significance. Therefore, A. octodonta may function as habitat "islands" providing a unique set of environmental conditions for luminous bacteria colonization, quite different from those already recorded from the plankton for other Vibrio species.

Control of mRNA processing and decay in prokaryotes.

Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA. As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5' to 3' directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease'. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.

In vivo analysis of the mechanisms responsible for strong transcriptional polarity in a "sense" mutant within an intercistronic region.

We have studied a very unusual strong polar mutant in the intercistronic barrier between the second (hisD) and third (hisC) cistrons of the histidine operon of Salmonella typhimurium to obtain further insights into the molecular mechanisms leading to transcription termination within cistrons. We have performed a detailed transcriptional analysis in vivo and have found that the his mRNA in this polar mutant is reduced in size as a result of premature termination of transcription at a cryptic Rho-dependent site within the proximal region of the hisC cistron.

Features of the rho-dependent transcription termination polar element within the hisG cistron of Salmonella typhimurium.

Previous genetic analysis showed that the polar effects of mutations in the hisG cistron of Salmonella typhimurium are dependent on the presence of a single putative transcription termination element within the hisG gene. In fact, all proximal mutations causing translation termination are strongly polar, whereas distal ones are not. The element was mapped by isolating mutations able to relieve the polar phenotype, and they were found to be small deletions in the region downstream of the translational stop codon (M. S. Ciampi and J. R. Roth, Genetics 118:193-202, 1988). In this study, we analyzed the his-specific RNAs synthesized in vivo in different strains harboring the polar frameshift hisG2148 mutation. The nature of the polarity effects is clearly transcriptional, since shorter RNA molecules were produced. When the hisG2148 mutation was transferred in a rho background or in strains harboring the small distal deletions, an increase in readthrough transcription was observed. The transcriptional termination element was characterized in more detail by performing high-resolution S1 nuclease mapping experiments. This analysis showed that (i) termination or exonucleolytic degradation following termination produced transcripts with heterogeneous 3' ends; (ii) this process is dependent on the transcription termination factor Rho, since relief of termination occurs in a rho background; and (iii) the element appears to function as a transcription terminator, at least to some extent, even in the course of active translation of the hisG cistron.

The Mu1 transposable element of maize contains two promoter signals recognized by the Escherichia coli RNA polymerase.

The galactokinase (GalK) expression plasmid vector system pKO-1 has been used to screen for promoter elements in the maize transposable element Mu1 that function in Escherichia coli. Two transcriptional start points, named S1 and S2, were identified, which are located in the two direct repeats of the transposable element. This paper demonstrates that sequence elements exist in a plant transposable element which function as prokaryotic promotors.

Nucleotide sequences of the genes regulating O-polysaccharide antigen chain length (rol) from Escherichia coli and Salmonella typhimurium: protein homology and functional complementation.

In this article, we report on the nucleotide sequences of the rol genes of Escherichia coli O75 and Salmonella typhimurium LT2. The rol gene in E. coli was previously shown to encode a 36-kDa protein that regulates size distribution of the O-antigen moiety of lipopolysaccharide. The E. coli and S. typhimurium rol gene sequences consist of 978 and 984 nucleotides, respectively. The homology between the nucleotide sequences of these two genes was found to be 68.9%. Both the E. coli rol and S. typhimurium rol genes are transcribed counter to the histidine operon and code for deduced polypeptides of 325 and 327 amino acids, respectively. The S. typhimurium rol gene was previously identified to encode a protein of unknown function and to share a transcription termination region with his. The homology between these deduced polypeptide sequences was observed to be 72%. A complementation test was performed in which the S. typhimurium rol gene was placed in trans with an E. coli plasmid (pRAB3) which encodes the O75 rfb gene cluster and not rol. The protein expressed from the S. typhimurium rol gene was found to regulate the distribution of the O75 O polysaccharide on the lipopolysaccharide of the host strain, E. coli S phi 874. The mechanism of Rol action may be independent of O antigen subunit structure, and its presence may be conserved in members of the family Enterobacteriaceae and other gram-negative bacilli that express O polysaccharides on their surface membrane.

A cytosine- over guanosine-rich sequence in RNA activates rho-dependent transcription termination.

We have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho factor-mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho-mediated mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.

A consensus motif common to all Rho-dependent prokaryotic transcription terminators.

We have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his-specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho-dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far.

Nucleotide sequence of the Escherichia coli hisD gene and of the Escherichia coli and Salmonella typhimurium hisIE region.

In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.

Intracistronic transcription termination in polysialyltransferase gene (siaD ) affects phase variation in Neisseria meningitidis.

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/-1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho-dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re-expression (off-on) without affecting the rates of loss of capsule expression (on-off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD-ermC' gene fusions in wild type and Rho-defective Escherichia coli strains. These studies demonstrated that in the Rho-defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off-on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD-ermC' gene fusions in a DNA mismatch-defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.