The cloning and characterization of a second alpha-amylase of A. hydrophila JMP636

AIMS: The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA.METHODS AND RESULTS: The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases.CONCLUSIONS: AmyB has been isolated, characterized and then compared to AmyA.SIGNIFICANCE AND IMPACT OF THE STUDY: The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.     

The gene encoding a periplasmic deoxyribonuclease from Aeromonas hydrophila

A gene encoding a deoxyribonuclease, dnsH, was cloned from Aeromonas hydrophila JMP636. The predicted mature protein was very similar to the previously described extracellular Dns from this organism and an N-terminal region corresponding to a large putative signal sequence was predicted for the JMP636 protein. Inactivation of dnsII demonstrated that the DnsH protein was not present extracellularly in this strain. As DnsH degraded plasmid DNA and was believed to have a periplasmic location, a dnsH mutant was constructed to determine whether electroporation of A. hydrophila with plasmid DNA could be achieved. No transformants were detected. From SDS-PAGE studies, at least two additional DNases remain to be characterised from A. hydrophila JMP636.     

Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila.

The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.     

Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method

Aims:  Aeromonas hydrophila is recognized as a human pathogen following wound exposure or ingestion of contaminated water and food. For rapid identification of this bacterium, a TaqMan-based real-time PCR assay has been developed.
Methods and Results:  Primers and probes that target specific sequences of the 16S rRNA gene and cytolytic enterotoxin gene ( aerA ) were combined in a duplex assay. Presence and size of PCR products were confirmed with microchannel fluidics electrophoresis analysis. After validation, using type strain CIP7614T DNA, the PCR assay was tested on 12 positive and negative controls. Twenty-one Aeromonas strains were isolated from environmental samples and were identified with biochemical tests as Aer. sobria , Aer. caviae and Aer. hydrophila . Only Aer. hydrophila strains tested positive by PCR assay.
Conclusions:  The PCR developed here was successfully applied for the identification of Aer. hydrophila from reference, clinical and environmental samples and showed a high discrimination between Aer. hydrophila and other Aeromonas species.
Significance and Impact of the Study:  This molecular method is convenient, rapid (2·5 h vs 24 h), specific to identify Aer. hydrophila and usable for diagnosis in medical and veterinary laboratories.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

Detection of metallo-β-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims:  To determine the prevalence and expression of metallo-β-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil.
Methods and Results:  Eighty - seven Aeromonas isolates belonging to Aeromonas hydrophila ( n  =   41) and Aer. jandaei ( n  =   46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97·6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla _IMP , bla _VIM and bla _SPM-1 were not detected. On the other hand, production of MBL activity was detectable in 87·8% and 10·9% of the cphA -positive Aer. hydrophila and Aer. jandaei isolates respectively.
Conclusions:  Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity.
Significance and Impact of the Study:  These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.     

Production of a monoclonal antibody against Aeromonas hydrophila and its application to bacterial identification

AIMS: to develop a monoclonal antibody (MAb) for the rapid detection of Aeromonas hydrophila in human faeces. METHODS AND RESULTS: A monoclonal antibody with strong specificity against Aer. hydrophila was obtained by the fusion of myeloma cells and splenocytes of a mouse immunized with vegetative cells of Aer. hydrophila ATCC 7966, followed by a two-step selection against other species of the genera. ELISA analyses revealed that MAb 5F3 strongly reacts with all the Aer. hydrophila strains evaluated, showing a just basal reactivity against other species of the genera, especially Aer. sobria and Aer. caviae. CONCLUSIONS: MAb 5F3 was characterized as an IgG that recognized a polypeptide of approximately 110 kDa. SIGNIFICANCE AND IMPACT OF THE STUDY: This MAb could be used to detect Aer. hydrophila in human stool samples.     

Characterization of Escherichia coli adenylate cyclase mutants with modified regulation

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.     

Effects of cultural conditions on protease production by Aeromonas hydrophila.

Production of extracellular proteolytic activity by Aeromonas hydrophila was influenced by temperature, pH, and aeration. Conditions which produced maximal growth also resulted in maximal protease production. Enzyme production appeared to be modulated by an inducer catabolite repression system whereby NH4+ and glucose repressed enzyme production and complex nitrogen and nonglucose, carbon energy sources promoted it. Under nutritional stress, protease production was high, despite poor growth.     

Effects of cultural conditions on protease production by Aeromonas hydrophila

Production of extracellular proteolytic activity by Aeromonas hydrophila was influenced by temperature, pH, and aeration. Conditions which produced maximal growth also resulted in maximal protease production. Enzyme production appeared to be modulated by an inducer catabolite repression system whereby NH4+ and glucose repressed enzyme production and complex nitrogen and nonglucose, carbon energy sources promoted it. Under nutritional stress, protease production was high, despite poor growth.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Cloning and expression of a chitinase gene from Aeromonas hydrophila in Escherichia coli.

An extracellular secreted chitinase gene from Aeromonas hydrophila was cloned in Escherichia coli, and the gene product was detected in the culture medium. Like the natural chitinase protein, the excreted chitinase had a molecular weight of approximately 85,000 and was subject to catabolite repression by glucose.     

Phenotypic study by numerical taxonomy of strains belonging to the genus Aeromonas

AIMS: This study was undertaken to cluster and identify a large collection of Aeromonas strains. METHODS AND RESULTS: Numerical taxonomy was used to analyse phenotypic data obtained on 54 new isolates taken from water, fish, snails, sputum and 99 type and reference strains. Each strain was tested for 121 characters but only the data for 71 were analysed using the 'SSM' and 'SJ' coefficients, and the UPGMA clustering algorithm. At SJ values of > or = 81.6% the strains clustered into 22 phenons which were identified as Aer. jandaei, Aer. hydrophila, Aer. encheleia, Aer. veronii biogroup veronii, Aer. trota, Aer. caviae, Aer. eucrenophila, Aer. ichthiosmia, Aer. sobria, Aer. allosaccharophila, Aer. media, Aer. schubertii and Aer. salmonicida. The species Aer. veronii biogroup sobria was represented by several clusters which formed two phenotypic cores, the first related to reference strain CECT 4246 and the second related to CECT 4835. A good correlation was generally observed among this phenotypic clustering and previous genomic and phylogenetic data. In addition, three new phenotypic groups were found, which may represent new Aeromonas species. CONCLUSIONS: The phenetic approach was found to be a necessary tool to delimitate and identify the Aeromonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable traits for identifying Aeromonas as well as the possible existence of new Aeromonas species or biotypes are indicated.     

Occurrence, diversity and pathogenicity of mesophilic Aeromonas in estuarine waters of the Italian coast of the Adriatic Sea

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae , 46 (22%) as Aer. sobria , 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii . Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

Regulatory exaptation of the catabolite repression protein (Crp)-cAMP system in Pseudomonas putida

The genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaA(P. putida) gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaA(P. putida) was poorly translated or rendered non-functional in the heterologous host. Yet, generation of cAMP by CyaA(P. putida) could be verified by expressing the cyaA(P. putida) gene in a hypersensitive E. coli strain. On the other hand, the crp(P. putida) gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress-endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp-cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co-opted for a new use.     

Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP

CRP—cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene ( crp* ). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of β-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of β-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS—PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp * mRNA levels. The level of CRP* protein correlates with β-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp * expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya⁻ crp * cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.