Amanda E. Goodman

Multiple genes involved in chitin degradation from the marine bacterium Pseudoalteromonas sp. strain S91.

A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chiC, with the same transcriptional direction, and two unlinked genes, chiP and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, ChiB and ChiC exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while ChiP and ChiQ belonged to family 20. ChiP and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; ChiB bound chitin but had no chitinase activity; ChiC possessed strong chitinase activity and also bound chitin. Production of ChiC in S91 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of ChiC proteins in a chitinase-negative mutant. In Escherichia coli, ChiC appeared to be expressed from its own promoter.

Carbon dioxide as a regulator of gene expression in microorganisms

CO2 regulates gene expression across a diverse group of microorganisms including fungi, and both photosynthetic and non photosynthetic bacteria. The processes that CO2 regulates are diverse. Several CO2-responsive random promoter lacZ fusions of unknown function have been isolated from a marine Synechococcus and a Pseudoalteromonas sp., highlighting the wide effect of CO2 control in these organisms. Regulatory proteins have been described that mediate the CO2 response at transcription level in Bacillus anthracis, the group A streptococci and two Rhodobacter spp.. These regulatory proteins include: AcpA and AtxA that are involved in CO2 control of B. anthracis capsule and toxin production; Mga that regulates surface associated virulence factors in the group A streptococci; and RegB/A, a two component signal transduction system that responds to environmental stimuli including CO2, to regulate photosynthetic apparatus and CO2 fixation enzyme synthesis in Rhodobacter spp..

A molecular phylogeny of the Australian monitor lizards (Squamata : Varanidae) inferred from mitochondrial DNA sequences

To date no complete phylogeny of all of the currently recognised Indo-Australian varanid species and subspecies has been published. This paper presents a comprehensive mitochondrial gene phylogeny of these lizards. A portion of the mitochondrial genome comprising part of the ND4 gene and three adjacent tRNA genes (hereafter referred to as ND4) was analysed alone and, for a subset of the taxa, combined with previously published mitochondrial data. Similar tree topologies were produced by both datasets although combining the data helped resolve some of the unresolved or weakly supported nodes in the ND4 analyses. The monophyly of the Indo-Australian group was strongly supported in all analyses. This group comprised three major lineages: the gouldii group, the Odatria group and the varius group. Mitochondrial ND4 nucleotide sequences were successfully amplified from all of the Indo-Australian monitor species and subspecies currently recognised and, as such, is the first comprehensive phylogenetic study of the Australian monitor lizards published. Analysis of the tempo of diversification and evolution of preferred habitat use identified six episodes of increased net speciation rate, with two closely adjacent episodes showing the highest rates of diversification and correlating with the appearance of all preferred habitat types. The comprehensive molecular phylogenetic framework will also be useful for the identification of varanid species and traded products derived from monitors and, as such, has important applications for wildlife management and conservation.

A molecular survey of a captive wallaby population for periodontopathogens and the co-incidence of Fusobacterium necrophorum subspecies necrophorum with periodontal diseases

Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.

Nitrogen regulates chitinase gene expression in a marine bacterium

Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by β-galactosidase assay of S91CX cultures containing different ammonium concentrations (NH4+; 0, 9.5 or 191 mM) and with different nitrogen sources (N-acetylglucosamine (GlcNAc) or glutamate (glt)). S91 chiA expression was found to depend on both the NH4+ concentration and source of nitrogen in marine minimal medium (MMM). Pseudoalteromonas sp. S91 and S91CX can use either GlcNAc or glt as a sole source of carbon in MMM containing a standard concentration of 9.5 mM NH4+. Adding excess NH4+, 20 times the standard concentration, to MMM significantly reduced chiA activity below that found in the presence of either GlcNAc or glt. When no NH4+ was added to MMM, S91CX was also able to use either GlcNAc or glt as a source of nitrogen; under these conditions chiA activity was significantly increased. Under all conditions tested, GlcNAc induced chiA activity significantly more than glt. Regulation of bacterial chitinases by nitrogen has not been previously reported. Transcriptional start point analysis of S91 chiA, using 5′RACE (ligation-anchored PCR), showed that during growth in MMM supplemented with (1) maltose (solely a carbon source for S91), chiA transcription occurred from only one putative σ70-dependent promoter; (2) the chitin monomer GlcNAc, transcription initiated from two putative σ54-dependent promoters and (3) glt, transcription initiated from all three putative promoters.

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster.

The chitinase gene cluster of the marine bacterium Pseudoalteromonas sp. S91, chiABC, which produces the major chitinases of this sp., was transcribed as an operon and from each individual gene. chiA, chiB and chiC were found to possess multiple transcriptional start points (TSPs), the use of which was determined by the nutrient regime used for S91 growth. In minimal medium containing glutamate, chiA, chiB and chiC each used 3, 1 and 1 TSP, respectively. Upon the addition of the chitin monomer N-acetylglucosamine, the number of chiA TSPs was unaffected. However, chiB used an additional 4 TSPs, and chiC used four new TSPs excluding the TSP used in glutamate only. In addition, the cluster was transcribed as an operon from TSP A1 of chiA. All TSPs were potentially associated with either a σ70- or σ54-dependent promoter. Under the growth conditions used, no TSPs were detected for chiB or chiC in S91CX, a chiA transposon mutant. The transcription of the S91 chiABC gene cluster produced at least four polycistronic mRNAs. In addition, the occurrence of operon transcription of chiABC, and identification of an additional 12 putative TSPs within the gene cluster, gave an indication that each gene appeared to be transcribed from more than one promoter region upstream of each in-frame translation start codon. Questions arose regarding the reason for this complexity of transcription within the gene cluster, leading to a re-evaluation of the Chi protein domains. By bioinformatic review, ChiA, ChiB and ChiC were found to potentially possess additional putative domains.

Nasal Colonization of Staphylococcus Spp among Captive and Free-Ranging Wallabies in South Australia

Nasal Colonization of Staphylococcus Spp among Captive and Free-Ranging Wallabies in South Australia Staphylococcal species diversity has been well studied with regard to antibiotic resistance in humans and animals of commercial or social value. However, studies of free-ranging wildlife and animals of conservation value are limited. In this study, multidrug resistant staphylococci were found exclusively in free-ranging wallabies indicating human activity and prior antibiotic exposure may not be significant contributing factors to the development of antibiotic resistance in staphylococci in animal reservoirs.

Characterisation of β-lactam resistance mediated by blaZ in staphylococci recovered from captive and free-ranging wallabies

Staphylococci are commensal organisms of animals, but some species are opportunistic pathogens that are resistant to almost all antimicrobial agents in clinical use. Bacterial resistance to β-lactam antimicrobial agents is widespread and has been investigated in species isolated from humans in addition to food production and companion animals. However, minimal progress has been made towards identifying reservoirs of β-lactam-resistant staphylococci in wildlife. This study was aimed at investigating and characterising β-lactamase resistance from staphylococci of wallaby origin. Staphylococci from free-ranging and captive wallabies were assessed for their phenotypic susceptibility to β-lactam antimicrobial agents prior to sequence analysis of their blaZ and blaR1 genes. Deduced amino acid sequences were classified according to the Ambler molecular characterisation method, assigned a protein signature type and compared with sequences generated from previous studies involving isolates from humans, cattle and companion animals. All BlaZ sequences identified in this study were assignable to a pre-existing β-lactamase class and protein signature type, including the more recently discovered protein signature type 12. Three major phylogenetic groups were resolved upon phylogenetic analysis against published BlaZ sequences. This study has found antibiotic-resistant staphylococci both in free-ranging and captive wallaby populations and these bacteria harbour blaZ variants that are different to those recovered from humans, cattle and companion animals. Further studies of staphylococci from non-traditional sources are required in order to enhance our knowledge of the epidemiology of antibiotic resistance genes.

Detection of Fusobacterium necrophorum Leukotoxin (lkta) Gene Sequence in the Oral Cavity of Captive Macropods

Fusobacterium necrophorum is an important aetiological agent of periodontal diseases (gingivitis and lumpy jaw) in captive macropods. The leukotoxin encoded by the lktA gene is a major virulence factor of F. necrophorum. In the present study, lktA gene sequences were detected by PCR in the oral cavity of 21/58 (36%) captive yellow footed rock wallabies and mainland tammar wallabies at two zoological parks in South Australia. This suggest that F. necrophorum encoded lktA may not be present in the oral cavity of all captive wallabies or that it is present at low levels, i.e. close to the limits of detection of conventional PCR. The lktA sequences had 98-100% homology to a sequence detected in ovine foot lesions infected with F. necrophorum. Cluster analysis of the partial lktA gene sequence revealed two clades of the lktA gene of F. necrophorum in macropods and calls for a detailed study using the whole gene sequence.

Plasmid Maintenance and Localisation of Vibrio sp. S141(p519ngfp) Cells within Monoculture and Mixed-species Biofilms

This study examined cell localisation and plasmid maintenance of Vibrio sp. S141(p519ngfp) cells which grew in, and detached from, monoculture and mixed-species marine biofilms under continuous flow conditions. Over the 48 h time course of the experiments, the broad host range IncQ RSF1010 derivative plasmid, p519 ngfp , was maintained in S141(p519ngfp) cells detaching from the biofilms irrespective of selection for the plasmid, the presence of another bacterial species, or the order of substratum colonisation. S141(p519ngfp) cell localisation within mixed-species biofilms was affected by the order, and length of time, of colonisation. When S141(p519ngfp) was the initial coloniser of the biofilm, the localisation of the majority of the plasmid-bearing cells near the substratum surface was not affected. When S141(p519ngfp) cells colonised a pre-existing Psychrobacter sp. SW5HR biofilm, or the two species simultaneously colonised the substratum, non plasmid-bearing cells initially dominated the substratum surface; as the time of S141(p519ngfp) colonisation increased, the plasmid-bearing cells appeared to displace the SW5HR cells from the surface. Since the p519ngfp plasmid was stably maintained in the Vibrio sp. S141 host forming biofilms over a 48 h period, GFP-producing S141 cells were able to be localised in mixed-species biofilms and were found to dominate the substratum surface by 48 h.