Background. Two non-tuberculous mycobacterial strains, UM_3 and UM_11, were isolated from the trunk wash of captive elephants in Malaysia. As they appeared to be identical phenotypes, they were investigated further by conventional and whole genome sequence-based methods of strain differentiation. Methods. Multiphasic investigations on the isolates included species identification with hsp65 PCR-sequencing, conventional biochemical tests, rapid biochemical profiling using API strips and the Biolog Phenotype Microarray analysis, protein profiling with liquid chromatography-mass spectrometry, repetitive sequence-based PCR typing and whole genome sequencing followed by phylogenomic analyses. Results. The isolates were shown to be possibly novel slow-growing schotochromogens with highly similar biological and genotypic characteristics. Both strains have a genome size of 5.2 Mbp, G+C content of 68.8%, one rRNA operon and 52 tRNAs each. They qualified for classification into the same species with their average nucleotide identity of 99.98% and tetranucleotide correlation coefficient of 0.99999. At the subspecies level, both strains showed 98.8% band similarity in the Diversilab automated repetitive sequence-based PCR typing system, 96.2% similarity in protein profiles obtained by liquid chromatography mass spectrometry, and a genomic distance that is close to zero in the phylogenomic tree constructed with conserved orthologs. Detailed epidemiological tracking revealed that the elephants shared a common habitat eight years apart, thus, strengthening the possibility of a clonal relationship between the two strains.
Biofilm formation and its molecular signaling in bacteria resistant to ionizing radiation is not fully understood. This study aimed to investigate the genetic variations and gene expression of biofilm in an ionizing radiation-resistant Bacillus subtilis in Ramsar. Direct sequencing and quantitative PCR were applied to determine nucleotide variations and gene expression profiles of tapA-sipW-tasA, sinR, sinI, ccpA, epsA-O, spoOB, spoOA, slrA, slrR, ymcA and abrB genes. RNAsnp-RNAfold and Phyre2 and the Swiss Model webserver were used to analyze the structural mRNA and protein respectively. At the molecular level, the tapA-sipW-tasA operon was significantly overexpressed and the expression of ccpA and slrR was significantly downregulated. The thermodynamic and ensemble diversity ratio of the tapA (G>C) gene showed the largest changes in RNA secondary structure. In addition, the largest protein pocket belonged to tapA (148.6 A03) compared to the normal structure (121.1 A03). A non-radiation Bacillus subtilis was served as a control group. These results support the hypothesis that the induction of robust biofilm formation is through the (tapA) operon signal in ionizing radiation-resistant B. subtilis and that genetic variation in tapA (G>C) was the major gene associated with diversity in robust biofilm formation.
Cell-cell interactions between genetically distinct bacteria, known as coaggregation, are important for the formation of mixed-species biofilms such as dental plaque. Interactions lead to gene regulation in the partner organisms that may be critical for adaptation and survival in mixed-species biofilms. Here, gene regulation responses to coaggregation between Streptococcus gordonii and Fusobacterium nucleatum were studied using dual RNA-Seq. Initially, S. gordonii was shown to coaggregate strongly with F. nucleatum in buffer or human saliva. Using confocal laser scanning microscopy and transmission electron microscopy, cells of different species were shown to be evenly distributed throughout the coaggregate and were closely associated with one another. This distribution was confirmed by serial block face sectioning scanning electron microscopy, which provided high resolution three-dimensional images of coaggregates. Cell-cell sensing responses were analysed 30 minutes after inducing coaggregation in human saliva. By comparison with monocultures, 16 genes were regulated following coaggregation in F. nucleatum whereas 119 genes were regulated in S. gordonii. In both species, genes involved in amino acid and carbohydrate metabolism were strongly affected by coaggregation. In particular, one 8-gene operon in F. nucleatum encoding sialic acid uptake and catabolism was up-regulated 2- to 5-fold following coaggregation. In S. gordonii, a gene cluster encoding functions for phosphotransferase system-mediated uptake of lactose and galactose was down-regulated up to 3-fold in response to coaggregation. The genes identified in this study may play key roles in the development of mixed-species communities and represent potential targets for approaches to control dental plaque accumulation.
Serratia sp. ATCC 39006 produces intracellular gas vesicles to enable upward flotation in water columns. It also uses flagellar rotation to swim through liquid and swarm across semi-solid surfaces. Flotation and motility can be co-regulated with production of a β-lactam antibiotic (carbapenem carboxylate) and a linear tripyrrole red antibiotic, prodigiosin. Production of gas vesicles, carbapenem and prodigiosin antibiotics, and motility are controlled by master transcriptional and post-transcriptional regulators, including the SmaI/SmaR-based quorum sensing system and the mRNA binding protein, RsmA. Recently, the ribose operon repressor, RbsR, was also defined as a pleiotropic regulator of flotation and virulence factor elaboration in this strain. Here, we report the discovery of a new global regulator (FloR; a DeoR family transcription factor) that modulates flotation through control of gas vesicle morphogenesis. The floR mutation is highly pleiotropic, down-regulating production of gas vesicles, carbapenem and prodigiosin antibiotics, and infection in Caenorhabditis elegans, but up-regulating flagellar motility. Detailed proteomic analysis using TMT peptide labelling and LC-MS/MS revealed that FloR is a physiological master regulator that operates through subordinate pleiotropic regulators including Rap, RpoS, RsmA, PigU, PstS and PigT.
Staphylococcus epidermidis is a pathogen associated with nosocomial infections whose medical importance has increased due to progressively invasive medical procedures. In this study, we characterized the molecular epidemiology of S. epidermidis strains circulating in our university hospital situated in Kuala Lumpur, Malaysia.
A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.
In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182 bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738 bp) and PHA synthase, class I (phaC(Co) gene, 1694 bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33 wt.% and 17 wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.
Pseudomonas sp. USM 4-55 is a locally isolated bacterium that possesses the ability to produce polyhydroxyalkanoates (PHA) consisting of both poly(3-hydroxybutyrate) [P(3HB)] homopolymer and medium-chain length (mcl) monomers (6 to 14 carbon atoms) when sugars or fatty acids are utilized as the sole carbon source. In this study, the P(3HB) biosynthesis operon carrying the phbC(Ps) P(3HB) synthase was successfully cloned and sequenced using a homologous probe. Three open reading frames encoding NADPH-dependent acetoacetyl-coenzyme A reductase (PhbB(Ps)), beta-ketothiolase (PhbA(Ps)) and P(3HB) synthase (PhbC(Ps)) were found in the phb operon. The genetic organization of phb operon showed a putative promoter region, followed by phbB(Ps)-phbA(Ps)-phbC(Ps). phbR(Ps)which encoded a putative transcriptional activator was located in the opposite orientation, upstream of phbBAC(Ps). Heterologous expression of pGEM''ABex harboring phbC(Ps) in Escherichia coli JM109 resulted in P(3HB) accumulation of up to 40% of dry cell weight (DCW).