Steve Petrovski

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to ‘Candidatus Accumulibacter phosphatis’ is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by ‘Candidatus Accumulibacter phosphatis’, and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

Characterization of the Genome of the Polyvalent Lytic Bacteriophage GTE2, Which Has Potential for Biocontrol of Gordonia-, Rhodococcus-, and Nocardia-Stabilized Foams in Activated Sludge Plants

ABSTRACT Hydrophobic Actinobacteria are commonly associated with the stabilization of foams in activated sludge systems. One possible attractive approach to control these foam-stabilizing organisms is the use of specific bacteriophages. We describe the genome characterization of a novel polyvalent DNA phage, GTE2, isolated from activated sludge. This phage is lytic for Gordonia terrae, Rhodococcus globerulus, Rhodococcus erythropolis, Rhodococcus erythropolis, Nocardia otitidiscaviarum, and Nocardia brasiliensis. Phage GTE2 belongs to the family Siphoviridae, possessing a characteristic icosahedral head encapsulating a double-stranded DNA linear genome (45,530 bp) having 10-bp 3′-protruding cohesive ends. The genome sequence is 98% unique at the DNA level and contains 57 putative genes. The genome can be divided into two components, where the first is modular and encodes phage structural proteins and lysis genes. The second is not modular, and the genes harbored there are involved in DNA replication, repair, and metabolism. Some have no known function. GTE2 shows promising results in controlling stable foam production by its host bacteria under laboratory conditions, suggesting that it may prove useful in the field as a biocontrol agent.

An examination of the mechanisms for stable foam formation in activated sludge systems

Screening pure cultures of 65 mycolic acid producing bacteria (Mycolata) isolated mainly from activated sludge with a laboratory based foaming test revealed that not all foamed under the conditions used. However, for most, the data were generally consistent with the flotation theory as an explanation for foaming. Thus a stable foam required three components, air bubbles, surfactants and hydrophobic cells. With non-hydrophobic cells, an unstable foam was generated, and in the absence of surfactants, cells formed a greasy surface scum. Addition of surfactant converted a scumming population into one forming a stable foam. The ability to generate a foam depended on a threshold cell number, which varied between individual isolates and reduced markedly in the presence of surfactant. Consequently, the concept of a universal threshold applicable to all foaming Mycolata is not supported by these data. The role of surfactants in foaming is poorly understood, but evidence is presented for the first time that surfactin synthesised by Bacillus subtilis may be important.

Genome Sequence and Characterization of the Tsukamurella Bacteriophage TPA2

ABSTRACT The formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizing Actinobacteria, among which are Tsukamurella spp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalent Siphoviridae family phage is lytic for most Tsukamurella species. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those of Mycobacterium phages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion on Tsukamurella phage evolution.

Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7

ABSTRACT Most activated sludge treatment plants suffer from the presence of foams on the surfaces of their aeration reactors. These are often stabilized by hydrophobic mycolic acid-synthesizing actinobacterial species. A polyvalent Siphoviridae phage, GTE7, which lysed several Gordonia and Nocardia species, is described here. Its genome has a modular structure similar to that described for Rhodococcus phage ReqiDocB7. In laboratory-scale experiments, we showed that GTE7 prevents stabilization of foams by these Gordonia and Nocardia species.

Small but Sufficient: the Rhodococcus Phage RRH1 Has the Smallest Known Siphoviridae Genome at 14.2 Kilobases

ABSTRACT Bacteriophages are considered to be the most abundant biological entities on the planet. The Siphoviridae are the most commonly encountered tailed phages and contain double-stranded DNA with an average genome size of ∼50 kb. This paper describes the isolation from four different activated sludge plants of the phage RRH1, which is polyvalent, lysing five Rhodococcus species. It has a capsid diameter of only ∼43 nm. Whole-genome sequencing of RRH1 revealed a novel circularly permuted DNA sequence (14,270 bp) carrying 20 putative open reading frames. The genome has a modular arrangement, as reported for those of most Siphoviridae phages, but appears to encode only structural proteins and carry a single lysis gene. All genes are transcribed in the same direction. RRH1 has the smallest genome yet of any described functional Siphoviridae phage. We demonstrate that lytic phage can be recovered from transforming naked DNA into its host bacterium, thus making it a potentially useful model for studying gene function in phages.

Tn502 and Tn512 Are res Site Hunters That Provide Evidence of Resolvase-Independent Transposition to Random Sites

In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.

Lysis to Kill: Evaluation of the Lytic Abilities, and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol.

Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.

Non‐target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non-target rRNA sequences. We show here that probe target sites containing single-base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non-target sites resulting from single-base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites.

The activated sludge bulking filament Eikelboom morphotype 0914 is a member of the Chloroflexi.

The filamentous bacterium Eikelboom morphotype type 0914 responsible for bulking in activated sludge plants is identified here for the first time as a member of the phylum Chloroflexi subgroup 1. Two FISH probes, CFX67a and CFX67b, targeting the 16S rRNA sequences of this filament morphotype were designed, validated and used successfully for its in situ identification. A survey of plants in eastern Australia with the CFX67a probe showed it targeted only the type 0914 morphotype that was common especially in long sludge age plants designed to remove phosphorus and nitrogen microbiologically, although being in very low abundance in many samples. Filaments responding to the CFX67b probe also exhibited the type 0914 morphology but were less frequent, although again occurring in similarly configured plants. All these filaments showed an uneven FISH signal suggesting their ribosomes are localized at the ends of their cells. Furthermore, some generated distinctive FISH signals in all biomass samples containing them, where only certain cells within any single trichome fluoresced with probes designed against different target sites. Helper probes for each of these were required before all cells fluoresced above the visual detection limits of FISH.