Draft Genome Sequences of Salmonella Lysozyme Gene Knockout Mutants

Abstract

Lysozyme enzymes hydrolyze the β-1,4-glycosidic bond in oligosaccharides. These enzymes are part of a broad group of glucoside hydrolases that are poorly characterized; however, they are important for growth and are being recognized as emerging virulence factors. This is the release of four lysozyme-encoding-gene-deletion mutants in Salmonella enterica serovar Typhimurium LT2.

Full text

Draft Genome Sequences of Salmonella
Lysozyme Gene Knockout Mutants
Narine Arabyan,
a,b
Bihua C. Huang,
a,b
Bart C. Weimer
a,b
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California,
Davis, Davis, California, USA
a
; 100K Pathogen Genome Project, UC Davis, Davis, California, USA
b
ABSTRACT Lysozyme enzymes hydrolyze the
␤
-1,4-glycosidic bond in oligosaccharides.
These enzymes are part of a broad group of glucoside hydrolases that are poorly
characterized; however, they are important for growth and are being recognized as
emerging virulence factors. This is the release of four lysozyme-encoding-gene-
deletion mutants in Salmonella enterica serovar Typhimurium LT2.
Lysozyme enzymes belong to the glucoside hydrolase 24 (GH24) family (1). GHs play
an important role during infection by altering the host glycan structure to gain
access to the host epithelial cells by binding to terminal monosaccharides to initiate
glycan degradation (2). Lysozyme enzymes recognize host GlcNAc containing glycans
in the form of N-glycans, O-glycans, glycolipids, glycoproteins, and glucosaminoglycans
during infection (3) for digestion, and hence may be new virulence factors due to
cleavage of the b-1,4-glycosidase bond. These GlcNAc molecules are linked to mono-
saccharides in the glycan via a
␤
-1,4-glycosidic bond (4) that can be cleaved by
enzymes from Salmonella with lysozyme activity during host association.
Lysozymes with
␤
-1,4-glycosidase activity are also involved during the secretion of
proteins, which is central for the virulence of all pathogenic bacteria (1). Gram-negative
organisms translocate proteins across the peptidoglycan that is composed of linear
chains of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), and the
alternating sugars are connected by
␤
-1,4-glycosidic bonds (5–7). The peptidoglycan
structure is a physical barrier for the assembly of macromolecular complexes and for
the transport of proteins. For this reason, all bacterial lysozymes degrade the pepti-
doglycan to allow the assembly of type III or type IV secretion systems essential for
virulence, flagella, or conjugation (8, 9). This remodeling creates gaps in the pepti-
doglycan necessary for the assembly of these macromolecular systems. Intracellular
pathogenic bacteria, such as Brucella abortus, use lysozyme during the early stages of
intracellular replication (8).
Four Salmonella enterica serovar Typhimurium LT2 lysozyme mutants (ΔSTM1028,
ΔSTM2612,ΔSTM2715.S, and ΔSTM3605 mutants) were constructed in the Weimer
laboratory (UC Davis, Davis, CA) (2), as described by Datsenko and Wanner (10). Cultures
were grown on 1.5% Luria-Bertani (LB) agar (Difco, Franklin Lakes, NJ) with 10
␮
g/ml
chloramphenicol at 37°C and lysed (11); genomic DNA (gDNA) was extracted (12) and
checked for quality (13); and sequencing libraries were constructed using the Kapa
HyperPlus kit, with enzymatic-based fragmentation (13), and indexed with Weimer 384
TS-LT DNA barcodes (Integrated DNA Technologies, Inc., Coralville, IA, USA) at 192
genomes/lane. The final libraries had average sizes of 350 to 450 bp (14, 15). All
genomes were sequenced on an Illumina HiSeq 4000 using PE150 (13, 16, 17) at the UC
Davis DNA Technologies Core (Davis, CA). Genome sequences were de novo assembled
using CLC Workbench version 6.5.1 (Qiagen), with default parameters.
This work was done as part of the 100K Pathogen Genome Project (http://www
.100kgenomes.org), which is a large-scale sequencing consortium that uses next-
generation sequencing methods to create genome databases for use in public health,
Received 24 April 2017 Accepted 28 April
2017 Published 8 June 2017
Citation Arabyan N, Huang BC, Weimer BC.
2017. Draft genome sequences of Salmonella
lysozyme gene knockout mutants. Genome
Announc 5:e00519-17. https://doi.org/10.1128/
genomeA.00519-17.
Copyright © 2017 Arabyan et al. This is an
open-access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.
Address correspondence to Bart C. Weimer,
bcweimer@ucdavis.edu.
PROKARYOTES
crossm
Volume 5 Issue 23 e00519-17 genomea.asm.org 1

food safety, and environmental science, where it is critical to capture genome diversity.
This project is focused on sequencing genomes of bacteria from the environment,
plants, animals, and humans worldwide, providing new insights into the genetic
diversity of pathogens and the microbiome.
Accession number(s). All sequences are publicly available and can be found at the
100K Project bioproject (NCBI PRJNA186441) in the Sequence Read Archive (http://
www.ncbi.nlm.nih.gov/sra), and genome assemblies can be found in NCBI GenBank
(see accession numbers in Table 1).
ACKNOWLEDGMENTS
B.C.W. is grateful for the funding that was contributed from Mars, Inc., NIH (grants
1R01HD065122-01A1 and NIH-U24-DK097154) and an Agilent Technologies Thought
Leader Award.
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TABLE 1 Salmonella enterica serovar Typhimurium LT2 deletion mutants with lysozyme activity
GenBank
accession no.
SRA
accession no.
Isolate
name
Gene
deleted Enzyme activity
No. of
contigs Coverage (ⴛ)
Total genome
size (bp)
No. of
CDSs
a
MZNN00000000 SRR5288766 BCW8410 ΔSTM1028 Lysozyme 68 156 4,894,775 4,816
MZNO00000000 SRR5288765 BCW8422 ΔSTM2612 Lysozyme 66 138 4,894,815 4,814
MZNP00000000 SRR5288764 BCW8423 ΔSTM2715.S Prophage lysozyme 67 138 4,894,604 4,807
MZYU00000000 SRR5288741 BCW8430 ΔSTM3605 Phage endolysin 59 79 4,893,277 4,803
a
CDSs, coding sequences.
Arabyan et al.
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