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Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

New evidence shows that host-microbiota crosstalk can be modulated via endogenous miRNAs. We have previously reported that miR-21 ablation protects against liver injury in cholestasis. In this study, we investigated the role of miR-21 in modulating the gut microbiota during cholestasis and its effects in liver dysfunction. Mice lacking miR-21 had reduced liver damage and were protected against small intestinal injury as well as from gut microbiota dysbiosis when subjected to bile duct ligation surgery. The unique microbiota profile of miR-21KO mice was characterized by an increase in Lactobacillus, a key microbiome genus for gut homeostasis. Interestingly, in vitro incubation of synthetic miR-21 directly reduced Lactobacillus load. Moreover, supplementation with Lactobacillus reuteri revealed reduced liver fibrosis in acute bile duct-ligated mice, mimicking the protective effects in miR-21 knockout mice. D-lactate, a main product of Lactobacillus, regulates gut homeostasis that may link with reduced liver fibrosis. Altogether, our results demonstrate that miR-21 promotes liver dysfunction through direct modulation of the gut microbiota and highlight the potential therapeutic effects of Lactobacillus supplementation in gut and liver homeostasis.

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2021-05-07T14:37:28Z No. of bitstreams: 1 ulfc126207_tm_Madalena_Marques.pdf: 1793493 bytes, checksum: b8bf8af99bea4c7d4fcea0aa77410c2a (MD5) Made available in DSpace on 2021-05-07T14:37:40Z (GMT). No. of bitstreams: 1 ulfc126207_tm_Madalena_Marques.pdf: 1793493 bytes, checksum: b8bf8af99bea4c7d4fcea0aa77410c2a (MD5) Previous issue date: 2020

SUMMARYMycobacterium tuberculosiscauses tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides – arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 Å resolution structure ofM. abscessusAftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 Å structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed inM. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

Babesia species, etiological agents of babesiosis, a recognized emerging tick-borne disease, are a significant animal and human health concern with a worldwide socio-economic impact. The development of genetic manipulation techniques, such as transfection technology, is pivotal to improve knowledge regarding the biology of these poorly studied parasites towards better disease control strategies. For Babesia ovis, responsible for ovine babesiosis, a tick-borne disease of small ruminants, these tools are not yet available. The present study was based on the existence of interchangeable cross-species functional promoters between Babesia species. Herein, we describe for the first time B. ovis transient transfection using two heterologous promoters, the ef-1α-B intergenic regions from B. bovis and B. ovata. Their ability to drive expression of a reporter luciferase in B. ovis supports their cross-species functionality. Also, the ef-1α-B promoter region from B. ovata resulted in statistically significantly higher luminescence values in comparison to the control, thus a possibly suitable promoter for stable gene expression. Evaluation of transfection efficiency using qPCR demonstrated that higher luminescence levels were due to promoter strength rather than a higher transfection efficiency. These findings represent a step forward in the development of methods for B. ovis genetic manipulation, an undoubtedly necessary tool to study this parasite basic biology, including its life cycle, the parasite interactions with host cells and virulence factors.

Mycobacteriophages are viruses that specifically infect mycobacteria, which ultimately culminate in host cell death. Dedicated enzymes targeting the complex mycobacterial cell envelope arrangement have been identified in mycobacteriophage genomes, thus being potential candidates as antibacterial agents. These comprise lipolytic enzymes that target the mycolic acid-containing outer membrane and peptidoglycan hydrolases responsive to the atypical mycobacterial peptidoglycan layer. In the recent years, a remarkable progress has been made, particularly on the comprehension of the mechanisms of bacteriophage lysis proteins activity and regulation. Notwithstanding, information about mycobacteriophages lysis strategies is limited and is mainly represented by the studies performed with mycobacteriophage Ms6. Since mycobacteriophages target a specific group of bacteria, which include Mycobacterium tuberculosis responsible for one of the leading causes of death worldwide, exploitation of the use of these lytic enzymes demands a special attention, as they may be an alternative to tackle multidrug resistant tuberculosis. This review focuses on the current knowledge of the function of lysis proteins encoded by mycobacteriophages and their potential applications, which may contribute to increasing the effectiveness of antimycobacterial therapy.

Bacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

Bacteriophages have developed multiple host cell lysis strategies to promote release of descendant virions from infected bacteria. This review is focused on the lysis mechanisms employed by tailed double-stranded DNA bacteriophages, where new developments have recently emerged. These phages seem to use a least common denominator to induce lysis, the so-called holin-endolysin dyad. Endolysins are cell wall-degrading enzymes whereas holins form 'holes' in the cytoplasmic membrane at a precise scheduled time. The latter function was long viewed as essential to provide a pathway for endolysin escape to the cell wall. However, recent studies have shown that phages can also exploit the host cell secretion machinery to deliver endolysins to their target and subvert the bacterial autolytic arsenal to effectively accomplish lysis. In these systems the membrane-depolarizing holin function still seems to be essential to activate secreted endolysins. New lysis players have also been uncovered that promote degradation of particular bacterial cell envelopes, such as that of mycobacteria.

Like most double-stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin-endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. In this study we investigated the lysis function of Gp1, which is encoded by the gp1 gene that lies immediately upstream of lysA. Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of Ms6 holin. Gp1 does not belong to the holin class of proteins, and we provide evidence that it shares several characteristics with molecular chaperones. We show that Gp1 interacts with LysA, and that this interaction is necessary for LysA delivery to its target. In addition, PhoA fusions showed that, in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1. We also show that Gp1 is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. We propose that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone-like protein, in a holin-independent manner.

Antibiotic-resistant infections and the stagnations in the development of new drugs have increased the demand for new therapeutic approaches against Mycobacterium tuberculosis. Innovative systems that are able to target and eradicate the bacteria in the infected host cells may represent a therapeutic breakthrough while avoiding latency. The development of nanosystems aiming a controlled and targeted intracellular drug release, have proved to increase cytosolic therapeutic concentration while reducing undesired side effects. This work’s main goal was to develop a host-directed strategy against mycobacterial infection through the design of a biocompatible nanocarrier for phage-derived protein delivery, using M. smegmatis as model. Since mycobacterial pathogenicity is strongly supported by the presence of lipids in the cell wall, their degradation induces bacterial destruction through cell wall hydrolysis. Phage-based lipolytic enzymes such as, LysB a mycolylarabinogalactan esterase, represent an appealing therapeutic approach. The herein proposed Ms6 LysB-containing lipid nanocarrier (SLN_LysB) explores the known advantages of nanomedicine-based systems for phagocytic cells selectively targeting thus allowing LysB intracellular accumulation and a more pronounced mycobacterial infection eradication. Adsorption efficiency value indicates the potential of this system as a protein nanocarrier. Moreover, promising outcomes were obtained in host-infected macrophages treated with SLN_LysB. The results show that the herein proposed strategy was more effective in inhibiting the growth of M. smegmatis than free LysB, which might be related to the nanocarrier internalization. Acting as effective protein nanocarriers, the protein-guided delivery in the infected phagocytic cells allows it to exert its hydrolytic action on the lipid layer of the Mycobacterium.

Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy. supported by TechnoPhage S.A. and Tecnifar; Foundation for Science and Technology (PTDC/SAUMIC/122816/2010 – ‘Biofilms in diabetic foot: microbial virulence characterization and cross-talk of major isolates’); FCT PhD fellowship (SFRH/BD/72872/2010); FCT ‘Ciência 2007’ programme info:eu-repo/semantics/publishedVersion

We have been witnessing an increased interest in bacteriophage studies focused on their use as antibacterial agents to fight pathogenic bacteria. This interest is a consequence of the phages' ability to lyse a bacterial host. Until recently, little was known about the mechanisms used by mycobacteriophages to induce lysis of their complex hosts. However, studies on Ms6-induced lysis have changed this scenario and provided new insights into the mechanisms of bacteriophage-induced lysis. Specific lysis protein genes have been identified in mycobacteriophage genomes, reflecting the particular mycobacterial cell envelope composition. These include enzymes that target mycolic acid–containing lipids and proteins that participate in the secretion of the phage endolysin, functioning as chaperone-like proteins. This chapter focuses on the current knowledge of mycobacteriophage-induced lysis, starting with an overview of phage lysis and basic features of the lysis players.

Purpose. O-(N-alkylamido)methyl esters of penicillin G were studied as a new class of prodrugs. Methods. Their hydrolysis in aqueous buffers containing 20 % (v/v) of acetonitrile was investigated by HPLC. Results. A U-shaped pH-rate profile was seen with a pH-independent process extending from pH ca. 2 to pH ca. 10. This pathway is characterised by kinetic data that are consistent with a uni-molecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Penicillin G and the corresponding amide are the ultimate products detected and isolated, indicating that β-lactam ring opening is much slower than ester hydrolysis. The O-(N-alkylamido)methyl esters of penicillin G displayed similar in vitro antibacterial activity to penicillin G itself. Conclusions. Compared to the penicillin G derivatives, the much higher stability of the O-(N-methylbenzamido)methyl benzoate, acetate and valproate esters (which gave rise to a Bronsted βlg value of ca. -1) suggests that tertiary N-acyloxymethylamides may be useful prodrugs for carboxylic acid drugs with pKa >4.

Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

Due to their bacterial lytic action, bacteriophage endolysins have recently gained great attention as a potential alternative to antibiotics in the combat of Gram-positive pathogenic bacteria, particularly those displaying multidrug resistance. However, large-scale production and purification of endolysins is frequently impaired due to their low solubility. In addition, a large number of endolysins appear to exhibit reduced lytic efficacy when compared with their action during phage infection. Here, we took advantage of the high solubility of two recently characterized enterococcal endolysins to construct chimeras targeting Staphylococcus aureus. The putative cell wall binding domain of these endolysins was substituted by that of a staphylococcal endolysin that showed poor solubility. Under appropriate conditions the resulting chimeras presented the high solubility of the parental enterococcal endolysins. In addition, they proved to be broadly active against a collection of the most relevant methicillin-resistant S. aureus epidemic clones and against other Gram-positive pathogens. Thus, fusion of endolysin domains of heterologous origin seems to be a suitable approach to design new potent endolysins with changed and/or extended lytic spectrum that are amenable to large-scale production.

Cryptolepine is the main alkaloid of Cryptolepis sanguinolenta (Lindl.) Schlechter, a plant used in traditional medicine in West Africa. The minimal inhibitory concentrations (MICs) of cryptolepine, ethanol and aqueous extracts of Cryptolepis sanguinolenta root were determined for 65 strains of Campylobacter jejuni, 41 strains of Campylobacter coli isolated from sporadic cases of gastroenteritis in Portugal and 86 strains of Vibrio cholerae isolated from patients with enteric infections in Angola, Brazil and Portugal. The ethanol extract activity against Campylobacter strains (MIC90% = 25 microg/ml) is higher than that of co-trimoxazole and sutfamethoxazole and Campvlobacter strains susceptibility for cryptoleprne (MIC90% = 12.5 microg/ml) is equal for ampicillin. The ethanot extract and cryptolepine show some activity against the Vibrio cholerae strains, although their activities are tower than that of tetracycline. lhe results suggest that these roots could be a therapeutic alternative for bacterial etiologic diarrhoea in West Africa., The authors express their thanks to JNICT Portugal for the financial support through the PMCT/C/SAU/749/90 project.

The intermolecular interactions of the mycobacteriophage Ms6 secretion chaperone with endolysin were characterized. The 384-amino-acid lysin (lysin 384 )-binding domain was found to encompass the N-terminal region of Gp1, which is also essential for a lysis phenotype in Escherichia coli . In addition, a GXXXG-like motif involved in Gp1 homo-oligomerization was identified within the C-terminal region.

Mycobacteriophages are dsDNA viruses that infect mycobacterial hosts. The mycobacteriophage Ms6 accomplishes lysis by producing two cell wall hydrolytic enzymes, Lysin A (LysA) that possesses a central peptidoglycan recognition protein (PGRP) super-family conserved domain with the amidase catalytic site, that cleaves the amide bond between the N-acetylmuramic acid and L-alanine residues in the oligopeptide crosslinking chains of the peptidoglycan and Lysin B (LysB) a mycolylarabinogalactan esterase that hydrolyzes the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex. Examination of the endolysin (lysA) DNA sequence revealed the existence of an embedded gene (lysA(241)) encoded in the same reading frame and preceded by a consensus ribosome-binding site. In the present work we show that, even though lysA is essential for Ms6 viability, phage mutants that express only the longer (Lysin(384)) or the shorter (Lysin(241)) endolysin are viable, but defective in the normal timing, progression and completion of host cell lysis. In addition, both endolysins have peptidoglycan hydrolase activity and demonstrated broad growth inhibition activity against various gram-positive bacteria and mycobacteria.

The mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) bacteriophage which, in addition to the predicted endolysin (LysA)-holin (Gp4) lysis system, encodes three additional proteins within its lysis module: Gp1, LysB, and Gp5. Ms6 Gp4 was previously described as a class II holin-like protein. By analysis of the amino acid sequence of Gp4, an N-terminal signal-arrest-release (SAR) domain was identified, followed by a typical transmembrane domain (TMD), features which have previously been observed for pinholins. A second putative holin gene ( gp5 ) encoding a protein with a predicted single TMD at the N-terminal region was identified at the end of the Ms6 lytic operon. Neither the putative class II holin nor the single TMD polypeptide could trigger lysis in pairwise combinations with the endolysin LysA in Escherichia coli . One-step growth curves and single-burst-size experiments of different Ms6 derivatives with deletions in different regions of the lysis operon demonstrated that the gene products of gp4 and gp5 , although nonessential for phage viability, appear to play a role in controlling the timing of lysis: an Ms6 mutant with a deletion of gp4 (Ms6 Δ gp4 ) caused slightly accelerated lysis, whereas an Ms6 Δ gp5 deletion mutant delayed lysis, which is consistent with holin function. Additionally, cross-linking experiments showed that Ms6 Gp4 and Gp5 oligomerize and that both proteins interact. Our results suggest that in Ms6 infection, the correct and programmed timing of lysis is achieved by the combined action of Gp4 and Gp5.

Like most double-stranded (ds) DNA phages, mycobacteriophage Ms6 uses the holin-endolysin system to achieve lysis of its host. In addition to endolysin (lysA) and holin (hol) genes, Ms6 encodes three accessory lysis proteins. In this study we investigated the lysis function of Gp1, which is encoded by the gp1 gene that lies immediately upstream of lysA. Escherichia coli lysis was observed after coexpression of LysA and Gp1 in the absence of Ms6 holin. Gp1 does not belong to the holin class of proteins, and we provide evidence that it shares several characteristics with molecular chaperones. We show that Gp1 interacts with LysA, and that this interaction is necessary for LysA delivery to its target. In addition, PhoA fusions showed that, in Mycobacterium smegmatis, LysA is exported to the extracytoplasmic environment in the presence of Gp1. We also show that Gp1 is necessary for efficient M. smegmatis lysis, as Ms6 gp1 deletion results in host lysis defects. We propose that delivery of Ms6 endolysin to the murein layer is assisted by Gp1, a chaperone-like protein, in a holin-independent manner.

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan–peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6′-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.

Several members of the Enterobacteriaceae, including Enterobacter spp., are naturally resistant to amoxicillin and cephalosporins. Enterobacter cloacae produces chromosomally encoded β-lactamases, also called cephalosporinases (1), and is a serious nosocomial pathogen, the third most prevalent bacterium isolated in intensive care settings (5, 8). We report here the study of a new chromosomal AmpC β-lactamase produced by E. cloacae FFUL2En isolated from the blood culture of a patient hospitalized in a medicine ward of Hospital de Santa Maria, Lisbon, Portugal. The antibiogram revealed resistance to aminopenicillins, aztreonam, and broad-spectrum cephalosporins, except imipenem, aminoglycosides, and quinolones. By isoelectrofocusing, the sonicate extracts expressed a pI of 8.68, suggesting the presence of a presumed AmpC enzyme. A total DNA preparation from E. cloacae FFUL2En was used in PCR experiments with two sets of primers, TN5 (5′-CGTTTGTCAGGCACAGTCAAATCCA) and TN4 (5′-TTACTGTAGCGCGTCGAGGATATGG) and the internal primers TN2 (5′-TTCCACTGCGGCTGCCAGT) and TN3 (5′-CGGATGAGGTCACGGATAACGCC), designed in accordance with consensus sequences from the ampC genes described for E. cloacae and available at GenBank. The amplicon with 1,234 bp was cloned into the SmaI site of the pBK-CMV vector (6) with a TOPO TA cloning kit, resulting in the plasmid p2En1. The β-lactam susceptibility pattern of Escherichia coli 2En1, harboring the recombinant plasmid p2En1, displayed cefoxitin, cefuroxime, ceftazidime, and piperacillin plus tazobactam MICs of >256 μg/ml and a cefepime MIC of 0.5 μg/ml. The MICs of cefotaxime and aztreonam were lower than those for the parental strain (Table ​(Table1).1). The E. coli 2En1 transformant showed the same pI as the parental strain (pI 8.68), and the substrate profile of the enzyme EcloFFUL2En was determined with the transformant crude enzymatic extract (7). The Vmax values indicate that cephalothin, with a Vmax of 3,000.1 μM/min, is hydrolyzed more quickly than cefoxitin (Vmax = 3.7 μM/min). Ceftazidime and cefotaxime are not hydrolyzed at detectable levels (Vmax =

A mycobacteriophage Ms6 strong promoter region ( P lys ) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem σ 70 -like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P lys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.

Terminalia macroptera Guill et Perr. (Combretaceae) is a medicinal plant used in Guinea-Bissau and other West African countries to treat infectious diseases. The ethanol extract from T. macroptera decorticated root and their liquid-liquid partition fractions, were screened for antimicrobial activity, by the twofold serial microdilution assay against seven reference bacterial strains and against Candida albicans. The extract and fractions showed some activity against at least one of the test microorganisms. The best results were obtained against Shigella dvsenteriae and Vibrio cholerae. The minimum inhibitory concentrations (MIC) of T. macroptera ethanol extract were also determined for about 100 clinical strains of Campylobacter sp., Escherichia coli, Salmonella sp., Shigella sp. and Vibrio cholerae. The ethanol extract activity against Campylobacter strains is similar to co-trimoxazole, higher than sulfamethoxazole but lower than tetracycline, erythromvcin, ampicilin and streptomycin. Ellagitannins are the major compounds in the extract and active fractions. The obtained results suggest a potential importance of this medicinal plant in the treatment of enteric diseases. particularly in Campylobacter infections., This work is part of the Project PMCT/C/SAU/ 749/90 financed by Junta Nacional de Investigação Científica e Tecnológica (JNICT), Portugal.

The ethanolic extracts of twelve plants selected through ethnomedical survey in Guinea-Bissau were investigated for their in vitro antimicrobial properties over ten bacteria and Candida albicans, using agar diffusion and dilution methods. All the tested extracts showed some activity against at least one of the bacteria. Most of the extracts (79%) showed activity against Staphylococcus aureus and only one (Cryptolepis sanguinolenta) against Escherichia coli. Cryptolepis sanguinolenta and Terminalia macroptera root extracts showed some activity against Candida albicans as well as showing an interesting profile of activity against most of the enteropathogen microorganisms. Inhibition zones against Staphylococcus aureus were localised on extract chromatograms by bioautographic techniques., This work is part of the Project PMCT/C/ SAU/749/90 financed by JNICT, Portugal.