Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

KARAKTERISASI BAKTERI DARI PERAKARAN Nepenthes mirabilis UNTUK PENGENDALIAN HAYATI Fusarium oxysporum

<em>Phytopathogenic fungi </em>Fusarium oxysporum<em> causes significant yield losses in various spices plants. The fungus can be controlled with numerous types of antagonistic bacteria. Th</em><em>is study aimed to determine the physiological characteristic and antagonistic properties of the bacterial from the roots of </em>Nepenthes mirabilis<em>, as a biological control </em><em>to </em>F. oxysforum<em>. The study was conducted at the Plant Protection Laboratory, Faculty of Agriculture, the University of Borneo, Tarakan</em><em>, from October to November 2017. Nutrient Agar medium was used to isolate antagonistic bacteria from the roots of </em>N. mirabilis<em>. Biosafety test against plants and mammals were conducted using hypersensitive and hemolysis </em><em>analysis. The bacterial isolates passed from those tests were characterized further for their phenotype and physiological properties as well as their ability to inhibit the growth of </em>F. oxysporum<em> in a dual culture test in vitro. The results showed that there were 10 out of</em><em>26 bacterial isolates originated from </em>N. mirabilis<em> roots that were safe for plants and mammals. </em><em>Physiological tests showed that four isolates could produce the proteolytic enzyme, five isolates produced the chitinolytic enzyme, six isolates were able to dissolve phosphate, and four isolates could produce HCN. Furthermore, three isolates (Mrb2, Mrb6, and Mrb16) showed inhibitory activity against </em>Fusarium<em> spp. There were differences in the phenotype character and physiological activity between the Mrb2, Mrb6, and Mrb16 isolates, but all three have the potential to inhibit </em>F. oxysporum<em>.</em>

Chitinolytic proteins secreted by Cellulosimicrobium sp. NTK2

ABSTRACT Cellulosimicrobium sp. NTK2 (NTK2 strain) was isolated as a chitinolytic bacterium from mature compost derived from chitinous waste. The growth of the NTK2 strain was enhanced by supplementation of the culture medium with 2% crystalline chitin. Approximately 70% of the supplemented crystalline chitin was degraded during cultivation. Whole genome analysis of the NTK2 strain identified eight chitinases and two chitin-binding proteins. The NTK2 strain secreted two bacterial extracellular solute-binding proteins, three family 18 glycosyl hydrolases and one lytic polysaccharide monooxygenase specifically in the presence of crystalline chitin. A chitinolytic enzyme with a molecular mass of 29 kDa on SDS-PAGE under native conditions was also secreted. This chitinolytic enzyme exhibited the largest band upon zymography but could not be identified. In an attempt to identify all the chitinases secreted by the NTK2 strain, we expressed recombinant versions of the proteins exhibiting chitinolytic activity in Escherichia coli. Our results suggest that the 29 kDa protein belonging to family 19 glycosyl hydrolase was expressed specifically in the presence of 2% crystalline chitin.

Aktivitas Bakteri Kitinolitik dari Cangkang Perna viridis sebagai Antifungi Phytophthora palmivora Penyebab Penyakit Busuk Buah Kakao (Theobroma cacao L.)

The emphasis on the growth of Phytophthora palmivora was important in order to reduce Pod Rot Disease of cacao (Theobrorna cacao L.) which could harm agriculture sector. Some bacteria had chitinolytic enzyme activity that is potentially used as an antifungal against Phytophthora palmivora, because the cell wall of the fungi composed of chitin. The purpose of this research was to know chitinolytic bacteria from Perna viridis shell which had higher activity of chitinase enzyme, the amount of chitinase enzyme activity of each selected isolate, and to know the effect of chitinolytic bacterial isolates from Perna viridis shell to reduce the growth of Phytophthora palmivora. The bacteria were isolated from Perna viridis shell at Depok Beach, Kretek, Bantul, Yogyakarta. This research was an explorative research which include bacterial characterization and experimental research which include antagonistic test of chitinolytic bacteria against Phytophthora palmivora. The chitinolytic bacteria was isolated using selective chitin agar medium by pour plate method and then screening the isolates that had chitinase enzyme activity by measuring the enzyme activity of each bacterial isolates by spectrophotometric method. Selected bacterial isolates were characterized by macroscopic, microscopic and physiological characters. The bacteria that had been selected tested for their ability to reduce the growth of Phytophthora palmivora by Kirby Bauer modification method. The result showed that there were 10 isolates that had chitinase enzyme activity which two selected isolates had the higher chitinase enzyme activity. There were 7D and 6B isolates. The isolate 7D had 1,258 u/ml chitinase enzyme activity and isolate 6B had 1,212 u/ml chitinase enzyme activity. The result of chitinolytic bacterial antagonist test on Phytophthora palmivora growth showed that both bacterial isolates were potential to antifungal Phytophthora palmivora and showed a real effect in inhibiting the growth of Phytophthora palmivora with significance value < 0,05.Keywords: Chitinolytic Bacteria, Perna viridis, Phytophthora palmivora

EKSPLORASI DAN UJI SENYAWA BIOAKTIF BAKTERI AGENSIA HAYATI UNTUK PENGENDALIAN PENYAKIT KRESEK PADA PADI

Exploration of bacterial biocontrol agent and its potential bioactive compound to control rice bacterial leaf blight. The research aims were to obtain bacterial isolates which were potential as biological control agent of X. oryzae pv. oryzae, the causal agent of rice bacterial blight and to assess the effectiveness of their bioactive compounds, and to identify of the potential isolates. The research steps included bacterial isolation, screening based on antibiosis activity and pathogenicity test, characterization based on chitinolytic enzyme production, siderophores, and phosphate dissolution test, effectiveness test of bioactive compounds and molecular identification of potential isolates. Out of 156 bacterial isolates from rice crop tested, 11 isolates showed to be non plant pathogenic and to have activity as biological agents against X. oryzae pv. oryzae pathotype III, IV and VIII. Further characterization of 11 isolates resulted in 2 isolates that showrd ability to produce chitinase (isolates T5-1118 and R7-1018), phosphatase (isolates T5-1105 and T6-1109), and siderophores (isolates T5-1118 and T6- 1109). The test of bioactive compound effectiveness of 4 isolates to the growth of X. oryzae pv. oryzae showed thatT5-1118, T5-1105, T6-1109 and R7-1018 have ability to inhibit X. oryzae pv. oryzae at 48 hours after inoculation of 66,61%, 62,4%, 23,97% and 12,40%, respectively. Identification of 4 bacterial biocontrol isolates with partial sequencing of 16S rRNA gene showed that those bacteria are close to Bacillus nealsonii strain F22 (R7-1018), Chromobacterium sp. MWU328 (T5-1118), Streptomyces sp. Antag 1 (T5-1105) and Kitasatospora nipponensis strains H2-4 (T6-1109).